14815, a human kinase family member and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 14815 nucleic acid molecules, which encode novel kinase family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 14815 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 14815 gene has been introduced or disrupted. The invention still further provides isolated 14815 proteins, fusion proteins, antigenic peptides and anti-14815 antibodies. Diagnostic and therapeutic methods utilizing compositions of the invention are also provided.

CROSS-REFERENCES TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/345,853, filed Jan. 2, 2002, the contents of whichare incorporated herein by this reference.

BACKGROUND OF THE INVENTION

[0002] The tight association of phosphate with a molecule, e.g., aprotein, has been known since the late nineteenth century. Since then, avariety of covalent linkages of phosphate to proteins have been found.The most common involve esterification of phosphate to serine,threonine, and tyrosine with smaller amounts being linked to lysine,arginine, histidine, aspartic acid, glutamic acid, and cysteine. Theoccurrence of phosphorylated molecules, e.g., proteins, implies theexistence of one or more kinases, e.g., protein kinases, capable ofphosphorylating various molecules, e.g., amino acid residues onproteins, and also of phosphatases, e.g., protein phosphatases, capableof hydrolyzing various phosphorylated molecules, e.g., phosphorylatedamino acid residues on proteins.

[0003] Protein kinases play critical roles in the regulation ofbiochemical and morphological changes associated with cellular growthand division (D'Urso et al. (1990) Science 250:786-791; Birchmeier etal. (1993) Bioessays 15:185-189). For example, these kinases have beenshown to participate in the transmission of signals from growth-factorreceptors (Sturgill et al. (1988) Nature 344:715-718; Gomez et al.(1991) Nature 353:170-173), control of entry of cells into mitosis(Nurse (1990) Nature 344:503-508; Mailer (1991) Curr. Opin. Cell Biol.3:269-275), and regulation of actin bundling (Husain-Chishti et al.(1988) Nature 334:718-721). Protein kinases serve as growth factorreceptors and signal transducers and have been implicated in cellulartransformation and malignancy (Hunter et al. (1992) Cell 70:375-387;Posada et al. (1992) Mol. Biol. Cell 3:583-592; Hunter et al. (1994)Cell 79:573-582). Alterations in kinase genes and their products canlead to deregulated cell proliferation, a hallmark of cancer. Modulationof these genes and their regulatory activities may permit the control oftumor cell proliferation and invasion.

[0004] Protein kinases can be divided into different groups based oneither amino acid sequence similarity or specificity for eitherserine/threonine or tyrosine residues. A small number ofdual-specificity kinases have also been described. Within the broadclassification, kinases can be further subdivided into families whosemembers share a higher degree of catalytic domain amino acid sequenceidentity and also have similar biochemical properties. Most proteinkinase family members also share structural features outside the kinasedomain that reflect their particular cellular roles. These includeregulatory domains that control kinase activity or interaction withother proteins (Hanks et al. (1988) Science 241:42-52).

SUMMARY OF THE INVENTION

[0005] The present invention is based, in part, on the discovery of anovel kinase family member, referred to herein as “14815”. The kinasemolecule of the invention shares characteristics with members of thetyrosine kinase family. The nucleotide sequence of a cDNA encoding 14815is shown in SEQ ID NO:1, and the amino acid sequence of a 14815polypeptide is shown in SEQ ID NO:2. In addition, the nucleotidesequence of the coding region is depicted in SEQ ID NO:3.

[0006] Accordingly, in one aspect, the invention features a nucleic acidmolecule which encodes a 14815 protein or polypeptide, e.g., abiologically active portion of the 14815 protein. In a preferredembodiment, the isolated nucleic acid molecule encodes a polypeptidehaving the amino acid sequence of SEQ ID NO:2. In other embodiments, theinvention provides isolated 14815 nucleic acid molecules having thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC AccessionNumber ______. In still other embodiments, the invention providesnucleic acid molecules that are substantially identical (e.g., naturallyoccurring allelic variants) to the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:3 or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC Accession Number ______. In otherembodiments, the invention provides a nucleic acid molecule whichhybridizes under a stringent hybridization condition as described hereinto a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:1, SEQ ID NO:3 or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC Accession Number ______, wherein the nucleicacid encodes a full length 14815 protein or an active fragment thereof.

[0007] In a related aspect, the invention further provides nucleic acidconstructs which include a 14815 nucleic acid molecule described herein.In certain embodiments, the nucleic acid molecules of the invention areoperatively linked to native or heterologous regulatory sequences. Alsoincluded are vectors and host cells containing the 14815 nucleic acidmolecules of the invention e.g., vectors and host cells suitable forproducing polypeptides.

[0008] In another related aspect, the invention provides nucleic acidfragments suitable as primers or hybridization probes for the detectionof 14815-encoding nucleic acids.

[0009] In still another related aspect, isolated nucleic acid moleculesthat are antisense to a 14815 encoding nucleic acid molecule areprovided.

[0010] In another aspect, the invention features 14815 polypeptides, andbiologically active or antigenic fragments thereof that are useful,e.g., as reagents or targets in assays applicable to treatment anddiagnosis of kinase-associated or other 14815-associated disorders. Inanother embodiment, the invention provides 14815 polypeptides having a14815 activity. Preferred polypeptides are 14815 proteins including atleast one protein kinase domain, at least one, two, three, preferablyfour armadillo/beta-catenin-like repeat domains, and, preferably, havinga 14815 activity, e.g., a 14815 activity as described herein.

[0011] In other embodiments, the invention provides 14815 polypeptides,e.g., a 14815 polypeptide having the amino acid sequence shown in SEQ IDNO:2 or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with ATCC Accession Number ______; an amino acidsequence that is substantially identical to the amino acid sequenceshown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with ATCC Accession Number ______; or anamino acid sequence encoded by a nucleic acid molecule having anucleotide sequence which hybridizes under a stringent hybridizationcondition as described herein to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 or the nucleotidesequence of the insert of the plasmid deposited with ATCC AccessionNumber ______, wherein the nucleic acid encodes a full length 14815protein or an active fragment thereof.

[0012] In a related aspect, the invention further provides nucleic acidconstructs which include a 14815 nucleic acid molecule described herein.

[0013] In a related aspect, the invention provides 14815 polypeptides orfragments operatively linked to non-14815 polypeptides to form fusionproteins.

[0014] In another aspect, the invention features antibodies andantigen-binding fragments thereof, that react with, or more preferablyspecifically or selectively bind 14815 polypeptides.

[0015] In another aspect, the invention provides methods of screeningfor compounds that modulate the expression or activity of the 14815polypeptides or nucleic acids.

[0016] In still another aspect, the invention provides a process formodulating 14815 polypeptide or nucleic acid expression or activity,e.g., using the compounds identified in the screens described herein. Incertain embodiments, the methods involve treatment of conditions relatedto aberrant activity or expression of the 14815 polypeptides or nucleicacids, such as conditions or disorders involving aberrant or deficientkinase function or expression. Examples of such disorders include, butare not limited to, cellular proliferative and/or differentiativedisorders, neurological disorders, inflammatory disorders, liverdisorders, apoptotic disorders, metabolic disorders and hormonaldisorders.

[0017] The invention also provides assays for determining the activityof or the presence or absence of 14815 polypeptides or nucleic acidmolecules in a biological sample, including for disease diagnosis.

[0018] In a further aspect, the invention provides assays fordetermining the presence or absence of a genetic alteration in a 14815polypeptide or nucleic acid molecule, including for disease diagnosis.

[0019] In another aspect, the invention features a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence. At least one address of the pluralityhas a capture probe that recognizes a 14815 molecule. In one embodiment,the capture probe is a nucleic acid, e.g., a probe complementary to a14815 nucleic acid sequence. In another embodiment, the capture probe isa polypeptide, e.g., an antibody specific for 14815 polypeptides. Alsofeatured is a method of analyzing a sample by contacting the sample tothe aforementioned array and detecting binding of the sample to thearray.

[0020] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWING

[0021]FIG. 1 depicts a hydropathy plot of human 14815. Relativelyhydrophobic residues are shown above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) are indicated by short vertical lines justbelow the hydropathy trace. The numbers corresponding to the amino acidsequence of human 14815 are indicated. Polypeptides of the inventioninclude fragments which include: all or part of a hydrophobic sequence,e.g., a sequence above the dashed line, e.g., the sequence from aboutamino acid 135 to 143, from about 225 to 237, and from about 634 to 642of SEQ ID NO:2; all or part of a hydrophilic sequence, e.g., a sequencebelow the dashed line, e.g., the sequence from about amino acid 51 to61, from about 90 to 104, and from about 802 to 815 of SEQ ID NO:2; asequence which includes a Cys, or a glycosylation site.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The human 14815 sequence (SEQ ID NO:1), which is approximately3919 nucleotides long including untranslated regions, contains apredicted methionine-initiated coding sequence of about 3348nucleotides, including the termination codon (nucleotides indicated ascoding of SEQ ID NO:1, SEQ ID NO:3). The coding sequence encodes a 1115amino acid protein (SEQ ID NO:2).

[0023] Human 14815 contains the following regions or other structuralfeatures (for general information regarding PFAM identifiers, PS prefixand PF prefix domain identification numbers, refer to Sonnhammer et al.(1997) Protein 28:405-420 or the Pfam website maintained in severallocations, e.g. by the Sanger Institute (pfam.sanger.ac.uk), WashingtonUniversity (pfam.wustl.edu), the Karolinska Institute (pfam.cgr.kr.se)or Institut de la National Recherche Agronomique (pfam.jouy.inra.fr) andthe ExPASy (Expert Protein Analysis System) proteomics server of theSwiss Institute of Bioinformatics (SIB), Geneva, Switzerland:

[0024] a protein kinase domain (PFAM Accession Number PF00069, SEQ IDNO:4) located at about amino acid residues 519 to 779 of SEQ ID NO:2;

[0025] four armadillo/beta-catenin-like repeat domains (PFAM AccessionNumber PF00514, SEQ ID NO:5) located at about amino acid residues 198 to238, 239 to 279, 280 to 320, and 401 to 448 of SEQ ID NO:2;

[0026] one protein kinases ATP-binding region signature (PrositePS00107, SEQ ID NO:6) located at about amino acids 525 to 548 of SEQ IDNO:2;

[0027] one tyrosine protein kinase active site signature (PrositePS00109, SEQ ID NO:7) located at about amino acids 651 to 663 of SEQ IDNO:2;

[0028] one coiled coil structure (PSORT, Nakai and Kanehisa (1992)Genomics 14:897-911), see the PSORT website maintained by the HumanGenome Center at the Institute of Medical Science in the University ofTokyo, psort.nibb.ac.jp.) located at about amino acids 472 to 500 of SEQID NO:2;

[0029] two 2^(nd) peroxisomal targeting signals (PTS2, SEQ ID NO:8,PSORT,, Nakai and Kanehisa (1992) Genomics 14:897-911), see the PSORTwebsite maintained by the Human Genome Center at the Institute ofMedical Science in the University of Tokyo, psort.nibb.ac.jp.) locatedat about amino acids 329 to 337 and 630 to 638 of SEQ ID NO:2;

[0030] one vacuolar targeting motif (VAC, PSORT, Nakai and Kanehisa(1992) Genomics 14:897-911), see the PSORT website maintained by theHuman Genome Center at the Institute of Medical Science in theUniversity of Tokyo, psort.nibb.ac.jp.) located at about amino acids 421to 424 of SEQ ID NO:2;

[0031] fourteen protein kinase C phosphorylation sites (Prosite PS00005)located at about amino acids 11 to 13, 15 to 17, 22 to 24, 40 to 42, 266to 268, 376 to 378, 567 to 569, 618 to 620, 755 to 757, 812 to 814, 852to 854, 964 to 966, 1098 to 1100, and 1113 to 1115 of SEQ ID NO:2;

[0032] fifteen casein kinase II phosphorylation sites (Prosite PS00006)located at about amino acids 22 to 25, 58 to 61, 235 to 238, 324 to 327,376 to 379, 479 to 482, 593 to 596, 618 to 621, 670 to 673, 696 to 699,898 to 901, 935 to 938, 1058 to 1061, 1067 to 1070, and 1082 to 1085 ofSEQ ID NO:2;

[0033] four cAMP/cGMP-dependent protein kinase phosphorylation sites(Prosite PS00004) located at about amino acids 267 to 270, 537 to 540,809 to 812, and 1026 to 1029 of SEQ ID NO:2;

[0034] seven N-glycosylation sites (Prosite PS00001) located at aboutamino acids 86 to 89, 96 to 99, 187 to 190, 401 to 404, 793 to 796, 911to 914, and 1095 to 1098 of SEQ ID NO:2;

[0035] one tyrosine kinase phosphorylation site (Prosite PS00007)located at about amino acids 740 to 746 of SEQ ID NO:2; and

[0036] eleven N-myristoylation sites (Prosite PS00008) located at aboutamino acids 67 to 72, 156 to 161, 209 to 214, 219 to 224, 228 to 233,355 to 360, 528 to 533, 609 to 614, 948 to 953, 960 to 965, and 1064 to1069 of SEQ ID NO:2.

[0037] A plasmid containing the nucleotide sequence encoding human14815, named Fbh14815FL, was deposited with American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209,on ______ and assigned Accession Number ______. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112.

[0038] The 14815 protein contains a significant number of structuralcharacteristics in common with members of the kinase family, inparticular, the tyrosine protein kinase family. The term “family” whenreferring to the protein and nucleic acid molecules of the inventionmeans two or more proteins or nucleic acid molecules having a commonstructural domain or motif and having sufficient amino acid ornucleotide sequence homology as defined herein. Such family members canbe naturally or non-naturally occurring and can be from either the sameor different species. For example, a family can contain a first proteinof human origin as well as other distinct proteins of human origin, oralternatively, can contain homologs of non-human origin, e.g., rat ormouse proteins. Members of a family also can have common functionalcharacteristics.

[0039] As used herein, the term “kinase” includes a protein orpolypeptide which is capable of modulating its own phosphorylation stateor the phosphorylation state of another molecule, e.g., protein orpolypeptide. Typically, a kinase obtains the phosphate group from atriphosphate molecule, e.g., a nucleotide (e.g., adenosine triphosphate(ATP)). Protein kinases can have a specificity for (i.e., a specificityto phosphorylate) serine or threonine residues, tyrosine residues, orserine, threonine and tyrosine residues, e.g., the dual specificitykinases. A kinase with specificity for tyrosine residues is referred toherein as a “tyrosine kinase.”

[0040] Members of a kinase family of proteins can be cytoplasmic,membrane-bound, or extracellular, and have a catalytic domain to performthe phosphorylation function. Regulation of the kinase activity canoccur by the association of the catalytic domain with another molecule,e.g. another protein, or by modification of the kinase domain tomodulate the kinase enzymatic activity. A kinase also can have one ormore additional domains to enable it to associate with a specificmolecule or location. Such an association can contribute to theregulation of the kinase or the regulatory function of the kinase.Examples of serine/threonine protein kinases include, but are notlimited to, cyclin-dependent kinases, protein kinase C (PKC), and caseinkinase II. Examples of tyrosine protein kinases include, but are notlimited to, members of the src subfamily, including some oncogenes,members of the c-src kinase subfamily, and members of the receptortyrosine kinase subfamily, including growth factor, e.g.platelet-derived growth factor and vascular endothelial growth factor,and hormone, e.g. insulin receptors. The 14815 polypeptide is a tyrosinekinase homologous to a partial amino acid sequence of a kinase familymember, SGK237 (SEQ ID NO:9, in WO 200166594). A GAP alignment of thisamino acid sequence to the 14815 polypeptide of SEQ ID NO:2 found 95.4%identity between the two sequences in the 854 amino acid region ofoverlap (as calculated in matblas from the blosum50matrix).

[0041] A 14815 polypeptide can include a “protein kinase domain” orregions homologous with a “protein kinase domain”. A 14815 polypeptidecan further include an “armadillo/beta-catenin-like repeat domain” orregions homologous with an “armadillo/beta-catenin-like repeat domain.”

[0042] As used herein, the term “protein kinase domain” includes anamino acid sequence of about 200 to 320 amino acid residues in lengthand having a bit score for the alignment of the sequence to the proteinkinase domain (HMM) of at least 200. Preferably a protein kinase domainmediates catalysis of protein phosphorylation and can mediate theinteraction with other domains, e.g. cyclin domains, kinase domains orankyrin repeat domains. Preferably, a protein kinase domain includes atleast about 220 to 300 amino acids, more preferably about 240 to 280amino acid residues, or about 255 to 265 amino acids and has a bit scorefor the alignment of the sequence to the protein kinase domain (HMM) ofat least 210, 220, 230 or greater.

[0043] The protein kinase domain can include a Prosite protein kinasesATP-binding signature sequence PS00107 (SEQ ID NO:6), or sequenceshomologous thereto at about amino acid residues 525 to 548 of SEQ IDNO:2. This sequence contains contains an active site lysine, e.g. K-548,which can bind the phosphate donor nucleotide, e.g., adenosinetriphosphate. The protein kinase domain also can include a Prositetyrosine protein kinases active site signature sequence PS00109 (SEQ IDNO:7), or sequences homologous thereto at about amino acid residues 651to 663 of SEQ ID NO:2. Within this sequence is an active site aspartateresidue, e.g. D-655.

[0044] As noted for the kinase signature sequence above, K-548 of SEQ IDNO:2 can be involved in ATP binding for the 14815 polypeptide.Experiments involving kinases typically use a kinase-dead mutant with asubstitution of the binding site lysine, e.g. K-548, to a differentamino acid residue, e.g. alanine by mutating nucleotides bases encodingthe lysine, e.g. nucleotides 1824 and 1825 of SEQ ID NO:1 from adenineto guanine and cytosine, respectively, so the codon for amino acidresidue 548 of SEQ ID NO:2 is GCG instead of the wild type AAG. Theresults of those experiments typically demonstrate loss of 14815 kinaseactivity with A instead of K at the binding site, e.g. residue 548 ofSEQ ID NO:2. Thus, further embodiments of the invention are asubstitution for K at residue 548 of SEQ ID NO:2, or a portion thereof,e.g. a polypeptide comprising a fragment of 14815, e.g. a polypeptidecomprising the kinase domain, and nucleic acid sequences encoding thesubstitution. Similarly, for the tyrosine kinase signature sequenceabove, D-655 of SEQ ID NO:2 can be an active site residue for the 14815polypeptide. Experiments involving kinases typically use a kinase-deadmutant with a substitution of the active site aspartate, e.g. D-655, toa different amino acid residue, e.g. alanine by mutating nucleotidesbases encoding the lysine, e.g. nucleotides 2146 and 2147 of SEQ ID NO:1from adenine and thymine to cytosine and guanine, respectively, so thecodon for amino acid residue 655 of SEQ ID NO:2 is GCG instead of thewild type GAT. The results of those experiments typically demonstrateloss of 14815 kinase activity with A instead of D at the active site,e.g. residue 655 of SEQ ID NO:2. Thus, further embodiments of theinvention are a substitution for D at residue 655 of SEQ ID NO:2, or aportion thereof, e.g. a polypeptide comprising a fragment of 14815, e.g.a polypeptide comprising the kinase domain, and nucleic acid sequencesencoding the substitution.

[0045] The protein kinase domain (HMM) has been assigned the PFAMAccession Number PF00069 (SEQ ID NO:4, (see the Pfam website maintainedin several locations, e.g. by the Sanger Institute (pfam.sanger.ac.uk),Washington University (pfam.wustl.edu), the Karolinska Institute(pfam.cgr.kr.se) or Institut de la National Recherche Agronomique(pfam.jouy.inra.fr)). The region containing the protein kinase domain(HMM) also has been assigned the SMART (modular architecture analysis)identifier “serine/threonine protein kinases, catalytic domain” (SM0220)and “tyrosine kinases, catalytic domain” (SM0219, smart.embl-heidelberg)and designated as located about amino acid residues 519 to 791 or 783,respectively, of SEQ ID NO:2. An alignment of the protein kinase domain(amino acids 519 to 779 of SEQ ID NO:2) of human 14815 proteins with thePfam protein kinase domain consensus amino acid sequence (SEQ ID NO:4)derived from a hidden Markov model yields a bit score of 236.7.

[0046] In a preferred embodiment, a 14815 polypeptide or protein has a“protein kinase domain” or a region which includes at least about 220 to300 amino acids, more preferably about 240 to 280 amino acid residues,or about 255 to 265 amino acid residues and has at least about 60%, 70%80% 90% 95%, 99%, or 100% homology with a “protein kinase domain,” e.g.,the protein kinase domain of human 14815 (e.g., residues 519 to 779 ofSEQ ID NO:2).

[0047] As used herein, the term “armadillo/beta-catenin-like repeatdomain” includes an amino acid sequence of about 20 to 60 amino acidresidues in length and having a bit score for the alignment of thesequence to the protein kinase domain (HMM) of at least 0.4. Thearmadillo/beta-catenin-like repeat domains typically have three alphahelices and the repeats can associate in a manner in which they form asuper-helix. Preferably, armadillo/beta-catenin-like repeat domainsmediate the interaction with another domain, e.g. a nucleoplasmindomain, an importin beta binding domain, a ras domain or a vinculindomain. These domains are found in proteins with diverse functions andlocations, from activities in the nucleus, cytoplasm (or both, dependingon the attachment of protein-modifying functional groups) and incytoplasmic-membrane anchoring structures. The structure of thesuper-helix can create a groove with a particular charge characteristicto mediate the association of the proteins containing the respectivedomains. Preferably, an armadillo/beta-catenin-like repeat domainincludes at least about 25 to 55 amino acids, more preferably about 30to 50 amino acid residues, or about 35 to 45 amino acids and has a bitscore for the alignment of the sequence to the protein kinase domain(HMM) of at least 0.6, 0.8, 1.0 or greater.

[0048] The armadillo/beta-catenin-like repeat domain (HMM) has beenassigned the PFAM Accession Number PF00514 (SEQ ID NO:5, (see the Pfamwebsite maintained in several locations, e.g. by the Sanger Institute(pfam.sanger.ac.uk), Washington University (pfam.wustl.edu), theKarolinska Institute (pfam.cgr.kr.se) or Institut de la NationalRecherche Agronomique (pfam.jouy.inra.fr)). The region containing theprotein kinase domain (HMM) also has been assigned the SMART (modulararchitecture analysis) identifier “armadillo/beta-catenin-like repeats”(SM0185, smart.embl-heidelberg) and designated as located about aminoacid residues 197 to 238, 278 to 320, and 401 to 448, of SEQ ID NO:2.Alignments of the armadillo/beta-catenin-like repeat domains (aminoacids 198 to 238, 239 to 279, 280 to 320, and 401 to 448 of SEQ ID NO:2)of human 14815 proteins with the Pfam armadillo/beta-catenin-like repeatdomain consensus amino acid sequence (SEQ ID NO:5) derived from a hiddenMarkov model yield bit scores of 15.5, 5.0, 6.3, and 1.1.

[0049] In a preferred embodiment, a 14815 polypeptide or protein has an“armadillo/beta-catenin-like repeat domain” or regions which include atleast about 25 to 55 amino acids, more preferably about 30 to 50 aminoacid residues, or about 35 to 45 amino acid residues and have at leastabout 60%, 70% 80% 90% 95%, 99%, or 100% homology with an“armadillo/beta-catenin-like repeat domain,” e.g., thearmadillo/beta-catenin-like repeat domains of human 14815 (e.g.,residues 198 to 238, 239 to 279, 280 to 320, and 401 to 448 of SEQ IDNO:2).

[0050] To identify the presence of a “protein kinase” domain or an“armadillo/beta-catenin-like repeat” domain in a 14815 protein sequence,and make the determination that a polypeptide or protein of interest hasa particular profile, the amino acid sequence of the protein can besearched against the Pfam database of HMMs (e.g., the Pfam database,release 2.1) using the default parameters (see the Pfam websitemaintained in several locations, e.g. by the Sanger Institute(pfam.sanger.ac.uk/Software/Pfam/HMM_search)). For example, the hmmsfprogram, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28:405-420 and a detailed descriptionof HMMs can be found, for example, in Gribskov et al. (1990) Meth.Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference. A search was performed against the HMMdatabase resulting in the identification of a “protein kinase domain”domain in the amino acid sequence of human 14815 at about residues 519to 779 of SEQ ID NO:2 and four “armadillo/beta-catenin-like repeatdomains” at about residues 198 to 238, 239 to 279, 280 to 320, and 401to 448 of SEQ ID NO:2.

[0051] An additional method to identify the presence of a “proteinkinase” or an “armadillo/beta-catenin-like repeat” domain in a 14815protein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a SMART database (Simple ModularArchitecture Research Tool, smart.embl-heidelberg) of HMMs as describedin Schultz et al. (1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultzet al. (2000) Nucl. Acids Res 28:231. The database contains domainsidentified by profiling with the hidden Markov models of the HMMer2search program (Durbin et al. (1998) Biological sequence analysis:probabilistic models of proteins and nucleic acids. Cambridge UniversityPress; see HMMer2 program documentation maintained by the WashingtonUniversity in St. Louis Mo., hmmer.wustl.edu/). The database also isextensively annotated and monitored by experts to enhance accuracy. Asearch was performed against the HMM database resulting in theidentification of a “serine/threonine protein kinases, catalytic domain”in the amino acid sequence of human 14815 at about residues 519 to 791of SEQ ID NO:2, a “tyrosine kinases, catalytic domain” in the amino acidsequence of human 14815 at about residues 519 to 783 of SEQ ID NO:2 andthree “armadillo/beta-catenin-like repeats” in the amino acid sequenceof human 14815 at about residues 197 to 238, 278 to 320, and 401 to 448,of SEQ ID NO:2.

[0052] A human 14815 protein also can include a coiled coil structure(PSORT, Nakai and Kanehisa (1992) Genomics 14:897-911). Coiled coilstructures are supercoiled helical domains responsible for theoligomerization of proteins. There is a characteristic heptad repeat(h-x-x-h-x-x-x)n in the coiled coil structures, where h representshydrophobic residues (Beck and Brodsky (1998) J. Struct. Biol.122:17-29). Coiled coil structures are found in a wide variety ofproteins, including cytoskeletal, nuclear, muscle, cell surface,extracellular, plasma, bacterial, and viral proteins and can be found inthe 14815 polypeptide at about amino acids 472 to 500 of SEQ ID NO:2.

[0053] A human 14815 protein can further include at least one,preferably two 2^(nd) peroxisomal targeting signals (PTS2, SEQ ID NO:8,PSORT, Nakai and Kanehisa (1992) Genomics 14:897-911). Peroxisomes aremembrane-bound organelles containing enzymes which perform a number offunctions, metabolic, catabolic and synthetic. Examples of functions atleast partially performed by peroxisomal enzymes include, but are notlimited to fatty acid metabolism, oxygen radical clearance andcholesterol synthesis. Peroxisomes are most abundant in liver, but manydisorders of peroxisomes are manifested as metabolic diseases orneurological diseases. The prediction of the peroxisome signal wasaccompanied by the secondary, seemingly conflicting predictions of amitochondrial presequence cleavage site, an endoplasmic reticulummembrane retention signal and a vacuolar targeting motif (VAC) by PSORT.Taken together, these results suggest that the 14815 kinase can have anintracellular location of a membrane-bound organelle, e.g. a peroxisome,the endoplasmic reticulum, a mitochondrion, or a vacuole. Preferably,the 14815 kinase has an intracellular location of a peroxisome.

[0054] A 14815 family member can include at least one protein kinasedomain; and at least one, two, three preferably fourarmadillo/beta-catenin-like repeat domains. A 14815 family member caninclude at least one protein kinases ATP-binding region signature(Prosite PS00107), at least one tyrosine protein kinase active sitesignature (Prosite PS00109), at least one coiled coil structure (PSORTcoiled coil), and at least one, preferably two 2^(nd) peroxisomaltargeting signals (PSORT, PTS2). Furthermore, a 14815 family member caninclude at least one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, preferably fourteen protein kinase Cphosphorylation sites (Prosite PS00005); at least one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,preferably fifteen casein kinase II phosphorylation sites (PrositePS00006); at least one, two, three, four, five, six, preferably sevenN-glycosylation sites (Prosite PS00001); at least one, two, three,preferably four cAMP/cGMP protein kinase phosphorylation sites (PrositePS00004); at least one tyrosine kinase phosphorylation site (PrositePS00007); and at least one, two, three, four, five, six, seven, eight,nine, ten preferably eleven N-myristoylation sites (Prosite PS00008).

[0055] As the 14815 polypeptides of the invention can modulate14815-mediated activities, they can be useful for developing noveldiagnostic and therapeutic agents for kinase-associated or other14815-associated disorders, as described below.

[0056] As used herein, a “kinase-associated activity” includes anactivity which involves phosphorylation of a protein, e.g.phosphorylation of a serine or threonine residue on a protein. Proteinkinases can play a role in signalling pathways associated with cellulargrowth. For example, protein kinases are involved in the regulation ofsignal transmission from cellular receptors, e.g., growth-factorreceptors; entry of cells into mitosis; and the regulation ofcytoskeleton function, e.g., actin bundling. These kinases can functionin these biological activities because of their ability to phosphorylatethemselves or other substrate molecules. Members of the kinase familycan play a role in cyclin-dependent kinase-neurodegenerative diseases(Smith et al. (2001) Cell Growth Differ. 12:277-83), myotonic dystrophy(Ueda et al. (2000) Prog. Histochem. Cytochem. 35:187-251), andPeutz-Jeghers syndrome (Westerman and Wilson (1999) Scand. J.Gastroenterol. Suppl. 230:64-70).

[0057] As used herein, a “14815 activity”, “biological activity of14815” or “functional activity of 14815”, refers to an activity exertedby a 14815 protein, polypeptide or nucleic acid molecule on e.g., a14815-responsive cell or on a 14815 substrate, e.g., a proteinsubstrate, as determined in vivo or in vitro. In one embodiment, a 14815activity is a direct activity, such as an association with a 14815target molecule. A “target molecule” or “binding partner” is a moleculewith which a 14815 protein binds or interacts in nature. In an exemplaryembodiment, 14815 is a kinase, e.g., a tyrosine protein kinase, and thusbinds to or interacts in nature with a molecule, e.g., a nucleotide(e.g. adenosine triphosphate) and a protein substrate, e.g. atyrosine-containing protein.

[0058] A 14815 activity can also be an indirect activity, e.g., acellular signaling activity mediated by interaction of the 14815 proteinwith a 14815 receptor. Based on the above-described sequence structuresand similarities to molecules of known function, the 14815 molecules ofthe present invention can have similar biological activities as kinasefamily members. For example, the 14815 proteins of the present inventioncan have one or more of the following activities: (1) the ability tobind a molecule, e.g., a nucleotide (e.g. adenosine triphosphate orguanine triphosphate); (2) the ability to bind a protein substrate, e.g.a tyrosine-containing protein or a serine or threonine-containingprotein; (3) the ability to catalyze the transfer of a functional group,e.g. a phosphate, from the nucleotide to the protein, e.g. to tyrosineresidue or a serine or threonine residue on the protein; (4) the abilityto bind a second protein, e.g. another 14815 molecule, a differentkinase, a cyclin, an ankyrin repeat-containing protein, nucleoplasmin,importin, a ras protein, or vinculin; (5) the ability to regulatetransmission of signals from cellular receptors, e.g., cell growthfactor receptors; (6) the ability to modulate the entry of cells, e.g.,precursor cells, into the cell cycle, e.g. mitosis or meiosis; (7) theability to modulate cellular differentiation; (8) the ability tomodulate cell death, e.g. apoptosis; and (9) the ability to regulatecytoskeleton function, e.g., actin bundling.

[0059] The 14815 molecules of the invention can modulate the activitiesof cells in tissues where they are expressed. For example, 14815 mRNA isexpressed in normal brain, normal liver, in breast tissue and primarybreast tumors, but much less in metastases from the primary tumor, inresting cells, but much less in these cells stimulated with inflammatorycytokines. Accordingly, the 14815 molecules of the invention can act astherapeutic or diagnostic agents for neurological disorders, hepaticdisorders, cellular proliferative and/or differentiative disorders, andinflammatory disorders.

[0060] The expression of 14815 mRNA was studied in further detail withregard to inflammation. Expression analysis of 14815 mRNA in cells ofthe immune system or involved in inflammatory responses showedregulation, with different amounts of 14815 expression in the resting orunstimulated cell, than the amounts in stimulated or activated cells ordiseased tissue. For example, 14815 expression in resting CD8+lymphocytes, e.g. cytotoxic T cells, is higher than in CD8+ lymphocytes,e.g. cytotoxic T cells, stimulated with anti-CD3 antibodies, so 14815can be involved in the growth, maintenance or activity of cytotoxic Tcells and play a role in allograft rejection. In another example, 14815expression in resting normal human lung fibroblasts is higher than innormal human lung fibroblasts stimulated with transforming growth factorbeta or tumor necrosis factor alpha, so 14815 can be involved in chronicconditions associated with respiratory inflammation, e.g. asthma, cysticfibrosis, chronic obstructive pulmonary disease and pulmonary fibrosis.In another example, 14815 expression in resting normal human dermalfibroblasts is higher than in normal human dermal fibroblasts stimulatedwith transforming growth factor beta or interleukin-1b, so 14815 can beinvolved in inflammatory skin conditions, such as psoriasis, allergicreactions, atopic dermatitis, and systemic sclerosis. In anotherexample, 14815 expression in resting fibroblast-like synoviocytes ishigher than in fibroblast-like synoviocytes stimulated with tumornecrosis factor alpha or interleukin-1, so 14815 can be involved ininflammatory joint conditions, such as arthritis, e.g. rheumatoidarthritis. In another example, 14815 expression in resting bronchialsmooth muscle cells is higher than in bronchial smooth muscle cellsstimulated with tumor necrosis factor alpha or interferon gamma, so14815 can play a role in asthma. The expression of 14815 shows differentlevels in resting normal human bronchial epithelial cells compared withinterleukin-4- or interleukin-13-stimulated normal human bronchialepithelial cells, so 14815 can have a role in Th2 responses, in theallergic response, in asthma, and/or in innate and adaptive immunity,e.g. in the response against nematodes or protozoa.

[0061] The 14815 molecules can be used to treat and/or diagnoseneurological disorders in part because 14815 mRNA is expressed in normalbrain and in part because aberrant or deficient function or expressionof peroxisomal proteins can result in disorders of the nervous system.Neurological disorders include disorders of the central nervous system(CNS) and the peripheral nervous system, e.g., cognitive andneurodegenerative disorders, Examples of neurological disorders include,but are not limited to, autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, Korsakoff'spsychosis, alcoholism, anxiety disorders, or phobic disorders; learningor memory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. Suchneurological disorders include, for example, disorders involvingneurons, and disorders involving glia, such as astrocytes,oligodendrocytes, ependymal cells, and microglia; cerebral edema, raisedintracranial pressure and herniation, and hydrocephalus; malformationsand developmental diseases, such as neural tube defects, forebrainanomalies, posterior fossa anomalies, and syringomyelia and hydromyelia;perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicella-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer's disease and Pick'sdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson's disease (paralysisagitans) and other Lewy diffuse body diseases, progressive supranuclearpalsy, corticobasal degenration, multiple system atrophy, includingstriatonigral degenration, Shy-Drager syndrome, and olivopontocerebellaratrophy, and Huntington's disease, senile dementia, Gilles de laTourette's syndrome, epilepsy, and Jakob-Creutzfieldt disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, including peroxisomal disorders (e.g., Zellwegersyndrome, adrenoleukodystrophy, Refsum's disease, or rhizomelicchondrodysplasia punctata), toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease. Further CNS-related disorders include, for example, thoselisted in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.

[0062] The 14815 molecules can be used to treat and/or diagnose liverdisorders in part because 14815 mRNA is expressed in normal liver and inpart because peroxisomes are abundant in the liver. Disorders associatedwith an accumulation in the liver of fibrous tissue, such as thatresulting from an imbalance between production and degradation of theextracellular matrix accompanied by the collapse and condensation ofpreexisting fibers. The methods described herein can be used to diagnoseor treat hepatocellular necrosis or injury induced by a wide variety ofagents including processes which disturb homeostasis, such as aninflammatory process, tissue damage resulting from toxic injury oraltered hepatic blood flow, and infections (e.g., bacterial, viral andparasitic). For example, the methods can be used for the early detectionof hepatic injury, such as portal hypertension or hepatic fibrosis. Inaddition, the methods can be employed to detect liver fibrosisattributed to inborn errors of metabolism, for example, fibrosisresulting from a storage disorder such as Gaucher's disease (lipidabnormalities) or a glycogen storage disease, A1-antitrypsin deficiency;a disorder mediating the accumulation (e.g., storage) of an exogenoussubstance, for example, hemochromatosis (iron-overload syndrome) andcopper storage diseases (Wilson's disease), disorders resulting in theaccumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia andgalactosemia) and peroxisomal disorders (e.g., Zellweger syndrome,adrenoleukodystrophy, Refsum's disease, or rhizomelic chondrodysplasiapunctata). Additionally, the methods described herein can be used forthe early detection and treatment of liver injury associated with theadministration of various chemicals or drugs, such as for example,methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine,tolbutamide or alcohol, or which represents a hepatic manifestation of avascular disorder such as obstruction of either the intrahepatic orextrahepatic bile flow or an alteration in hepatic circulationresulting, for example, from chronic heart failure, veno-occlusivedisease, portal vein thrombosis or Budd-Chiari syndrome.

[0063] The 14815 molecules can be used to treat and/or diagnose cellularproliferative and/or differentiative disorders in part because the 14815mRNA is expressed in the breast tissue and primary breast tumors, butmuch less in metastases from the primary tumor. Examples of cellularproliferative and/or differentiative disorders include cancer, e.g.,carcinoma, sarcoma, metastatic disorders or hematopoietic neoplasticdisorders, e.g., leukemias. A metastatic tumor can arise from amultitude of primary tumor types, including but not limited to those ofprostate, colon, lung, breast and liver origin.

[0064] As used herein, the term “cancer” (also used interchangeably withthe terms, “hyperproliferative” and “neoplastic”) refers to cells havingthe capacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. Cancerous diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, e.g., malignant tumor growth, or may becategorized as non-pathologic, i.e., a deviation from normal but notassociated with a disease state, e.g., cell proliferation associatedwith wound repair. The term is meant to include all types of cancerousgrowths or oncogenic processes, metastatic tissues or malignantlytransformed cells, tissues, or organs, irrespective of histopathologictype or stage of invasiveness. The term “cancer” includes malignanciesof the various organ systems, such as those affecting lung, breast,thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as wellas adenocarcinomas which include malignancies such as most coloncancers, precancerous conditions and associated diseases includingadenomatous polyposis of the colon and Turcot syndrome and hereditarydesmoid disease, renal-cell carcinoma, prostate cancer and/or testiculartumors, non-small cell carcinoma of the lung, cancer of the smallintestine and cancer of the esophagus. The term “carcinoma” is artrecognized and refers to malignancies of epithelial or endocrine tissuesincluding respiratory system carcinomas, gastrointestinal systemcarcinomas, genitourinary system carcinomas, testicular carcinomas,breast carcinomas, prostatic carcinomas, endocrine system carcinomas,and melanomas. Exemplary carcinomas include those forming from tissue ofthe cervix, lung, prostate, breast, head and neck, colon and ovary. Theterm “carcinoma” also includes carcinosarcomas, e.g., which includemalignant tumors composed of carcinomatous and sarcomatous tissues. An“adenocarcinoma” refers to a carcinoma derived from glandular tissue orin which the tumor cells form recognizable glandular structures. Theterm “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

[0065] The 14815 molecules of the invention can be used to monitor,treat and/or diagnose a variety of proliferative disorders. Suchdisorders include hematopoietic neoplastic disorders. As used herein,the term “hematopoietic neoplastic disorders” includes diseasesinvolving hyperplastic/neoplastic cells of hematopoietic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. Preferably, the diseases arise from poorly differentiated acuteleukemias, e.g., erythroblastic leukemia and acute megakaryoblasticleukemia. Additional exemplary myeloid disorders include, but are notlimited to, acute promyeloid leukemia (APML), acute myelogenous leukemia(AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus (1991)Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include,but are not limited to acute lymphoblastic leukemia (ALL) which includesB-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

[0066] Thus, the 14815 molecules can act as novel diagnostic targets andtherapeutic agents for controlling one or more cellular proliferativeand/or differentiative disorders or other kinase disorders. As usedherein, “kinase disorders” are diseases or disorders whose pathogenesisis caused by, is related to, or is associated with aberrant or deficientkinase protein function or expression. Examples of such disorders, e.g.,kinase-associated or other 14815-associated disorders, include but arenot limited to, cellular proliferative and/or differentiative disorders,as described above, as well as neurological disorders, liver disorders,apoptotic disorders, metabolic disorders and hormonal disorders.

[0067] The 14815 nucleic acid and protein of the invention can be usedto treat and/or diagnose immune e.g., inflammatory (e.g. respiratoryinflammatory), disorders in part because the 14815 mRNA expression isregulated by inflammatory cytokines in cells of the immune system, e.g.in CD8+ cells, and in cells which can be involved in inflammatorydiseases, e.g. in lung cells (e.g. fibroblasts, bronchial epithelialcells and bronchial smooth muscle cells), in skin cells (e.g. in dermalfibroblasts), and in joint cells (e.g. in fibroblast-like synoviocytes).Examples immune and inflammatory disorders or diseases include, but arenot limited to, autoimmune diseases (including, for example, diabetesmellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, inflammatorybowel disease, e.g. Crohn's disease and ulcerative colitis, aphthousulcer, iritis, conjunctivitis, keratoconjunctivitis, asthma, allergicasthma, chronic obstructive pulmonary disease, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitisposterior, and interstitial lung fibrosis), graft-versus-host disease,cases of transplantation, and allergy such as, atopic allergy.

[0068] The 14815 molecules can be used to treat apoptotic disorders inpart because kinase family members have the ability to modulate celldeath. Disorders involving aberrant or deficient apoptosis include, butare not limited to, autoimmune disorders such as systemic lupuserythematosus and immune-mediated glomerulonephritis; neoplasticdisorders such as follicular lymphoma and hormone dependent tumors ofthe breast, prostate gland and ovary; neurodegenerative disorders, suchas Alzheimer's disease, Huntington's disease, retinitis pigmentosa,amyotrophic lateral sclerosis, spinal muscular atrophy and Parkinson'sdisease; viral infections, such as those caused by herpesviruses,poxviruses and adenoviruses; blood disorders due to aberrant apoptoticactivity in the bone marrow, such as anemia associated with chronicdisease, i.e., aplastic anemia, chronic neutropenia and myelodysplasia;and tissue damage associated with myocardial infarctions and stroke.

[0069] The 14815 molecules can be used to treat metabolic disorders inpart because kinase family members have the ability to regulatetransmission of signals from cellular receptors. Diseases of metabolicimbalance include, but are not limited to, obesity, anorexia nervosa,cachexia, lipid disorders, and diabetes.

[0070] The 14815 molecules can be used to treat hormonal disorders inpart because kinase family members have the ability to regulatetransmission of signals from cellular receptors. Hormonal disordersinclude conditions or diseases in which the production and/or regulationof hormones in an organism is aberrant. Examples of such disorders anddiseases include type I and type II diabetes mellitus, pituitarydisorders (e.g., growth disorders), thyroid disorders (e.g.,hypothyroidism or hyperthyroidism), and reproductive or fertilitydisorders (e.g., disorders which affect the organs of the reproductivesystem, e.g., the prostate gland, the uterus, or the vagina; disorderswhich involve an imbalance in the levels of a reproductive hormone in asubject; disorders affecting the ability of a subject to reproduce; anddisorders affecting secondary sex characteristic development, e.g.,adrenal hyperplasia).

[0071] The 14815 protein, fragments thereof, and derivatives and othervariants of the sequence in SEQ ID NO:2 thereof are collectivelyreferred to as “polypeptides or proteins of the invention” or “14815polypeptides or proteins”. Nucleic acid molecules encoding suchpolypeptides or proteins are collectively referred to as “nucleic acidsof the invention” or “14815 nucleic acids.”

[0072] As used herein, the term “nucleic acid molecule” includes DNAmolecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., anmRNA) and analogs of the DNA or RNA generated, e.g., by the use ofnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

[0073] The term “isolated or purified nucleic acid molecule” includesnucleic acid molecules which are separated from other nucleic acidmolecules which are present in the natural source of the nucleic acid.For example, with regards to genomic DNA, the term “isolated” includesnucleic acid molecules which are separated from the chromosome withwhich the genomic DNA is naturally associated. Preferably, an “isolated”nucleic acid is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

[0074] As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology (1989) John Wiley & Sons, N.Y., 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are thepreferred conditions and the ones that should be used unless otherwisespecified.

[0075] As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., encodes a natural protein).

[0076] As used herein, the terms “gene” and “recombinant gene” refer tonucleic acid molecules which include an open reading frame encoding a14815 protein, preferably a mammalian 14815 protein, and can furtherinclude non-coding regulatory sequences, and introns.

[0077] An “isolated” or “purified” polypeptide or protein issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the protein is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. In one embodiment, the language “substantiallyfree” means preparation of 14815 protein having less than about 30%,20%, 10% and more preferably 5% (by dry weight), of non-14815 protein(also referred to herein as a “contaminating protein”), or of chemicalprecursors or non-14815 chemicals. When the 14815 protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation. The invention includes isolated or purified preparations ofat least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0078] A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence of 14815 (e.g., the sequence of SEQID NO:1 or 3) without abolishing or more preferably, withoutsubstantially altering a biological activity, whereas an “essential”amino acid residue results in such a change. For example, amino acidresidues that are conserved among the polypeptides of the presentinvention, e.g., those present in the protein kinase domain, arepredicted to be particularly unamenable to alteration.

[0079] A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a 14815protein is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a 14815 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for 14815 biological activity to identify mutants that retainactivity. Following mutagenesis of SEQ ID NO:1 or SEQ ID NO:3, theencoded protein can be expressed recombinantly and the activity of theprotein can be determined.

[0080] As used herein, a “biologically active portion” of a 14815protein includes a fragment of a 14815 protein which participates in aninteraction between a 14815 molecule and a non-14815 molecule.Biologically active portions of a 14815 protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the 14815 protein, e.g., the amino acidsequence shown in SEQ ID NO:2, which include fewer amino acids than thefull length 14815 protein, and exhibit at least one activity of a 14815protein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the 14815 protein, e.g.,phosphorylation of a protein, e.g. phosphorylation of a tyrosine residueon a protein, the binding of a nucleotide, e.g. ATP, to a 14815polypeptide, or the binding of a 14815 polypeptide to another protein,e.g. another 14815 protein, a different kinase, a cyclin, an ankyrinrepeat-containing protein, nucleoplasmin, importin, a ras protein, orvinculin. A biologically active portion of a 14815 protein can be apolypeptide which is, for example, 10, 25, 50, 100, 200 or more aminoacids in length. Biologically active portions of a 14815 protein can beused as targets for developing agents which modulate a 14815 mediatedactivity, e.g., phosphorylation of a protein, e.g. phosphorylation of atyrosine residue on a protein, the binding of a nucleotide, e.g. ATP, toa 14815 polypeptide, or the binding of a 14815 polypeptide to anotherprotein, e.g. another 14815 protein, a different kinase, a cyclin, anankyrin repeat-containing protein, nucleoplasmin, importin, a rasprotein, or vinculin.

[0081] Calculations of homology or sequence identity (the terms“homology” and “identity” are used interchangeably herein) betweensequences are performed as follows:

[0082] To determine the percent identity of two amino acid sequences, orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., the relative positions of the alignedsequences is set so areas of highest identity coincide, i.e. N-terminiof the sequences do not necessarily coincide). In a preferredembodiment, the length of a reference sequence aligned for comparisonpurposes is at least 30%, preferably at least 40%, more preferably atleast 50%, even more preferably at least 60%, and even more preferablyat least 70%, 80%, 90%, 100% of the length of the reference sequence(e.g., when aligning a second sequence to the 14815 amino acid sequenceof SEQ ID NO:2 having 1115 amino acid residues, at least 334, preferablyat least 446, more preferably at least 557, even more preferably atleast 669, and even more preferably at least 780, 892, or 1003 aminoacid residues are aligned). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”).

[0083] In one embodiment, e.g. in a global alignment, the calculation ofpercent identity accounts for the full length of the sequence of theinvention, e.g. the full length 14815 polypeptide of SEQ ID NO:2 (e.g.1115 amino acids in length). In another embodiment, e.g. in a gappedalignment, the calculation of percent identity accounts only for theportions of high identity contained by both sequences, with gapsintroduced in one or both sequences to ignore regions of low identity ormissing sequences. The percent identity between the two sequences in agapped alignment thus is a function of the number of identical positionsshared by the sequences, taking into account the number of gaps, and thelength of each gap, which need to be introduced for optimal alignment ofthe two sequences. For example, two sequences have 10 contiguousidentical amino acids, but one sequence additionally has 10 contiguousamino acids at its N-terminus. A global alignment of the 10 amino acidsequence with the 20 amino acid sequence, accounting for the 10additional amino acids, can give a result of 55% identity, but a gappedalignment of these sequences, discounting for the 10 additional aminoacids, can give a result of 100% identity.

[0084] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, e.g. for a global alignment, thepercent identity between two amino acid or nucleotide sequences can bedetermined using the algorithm of Meyers and Miller ((1989) CABIOS,4:11-17) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, Blosum 50 matrix, a gaplength penalty of 12 and a gap penalty of 4. By the global alignmentcalculation, 14815 polypeptides can have at least 60%, 65%, 75%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the aminoacid sequence of SEQ ID NO:2.

[0085] In another embodiment, e.g. for a gapped alignment, the percentidentity between two amino acid sequences is determined using theNeedleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithm which hasbeen incorporated into the GAP program in the GCG softwarepackage(available at the bioinformatics page of the website maintainedby Accelrys, Inc., San Diego, Calif., USA), using either a Blossum 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package (availableat the bioinformatics page of the website maintained by Accelrys, Inc.,San Diego, Calif., USA) using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if a molecule is within a sequence identity orhomology limitation of the invention) are a Blossum 62 scoring matrixwith a gap penalty of 12, a gap extend penalty of 4, and a frameshiftgap penalty of 5.

[0086] The nucleic acid and protein sequences described herein can beused as a “query sequence” to perform a search against public databasesto, for example, identify other family members or related sequences.Such searches can be performed using the NBLAST and XBLAST programs(version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength =12 to obtain nucleotide sequences homologous to 14815nucleic acid molecules of the invention. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to 14815 protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) can be used (accessible at the website maintained by NationalCenter for Biotechnology Information, Bethesda, Md., USA(ncbi.nlm.nih.gov)).

[0087] Particular 14815 polypeptides of the present invention have anamino acid sequence substantially identical to the amino acid sequenceof SEQ ID NO:2. In the context of an amino acid sequence, the term“substantially identical” is used herein to refer to a first amino acidthat contains a sufficient or minimum number of amino acid residues thatare i) identical to, or ii) conservative substitutions of aligned aminoacid residues in a second amino acid sequence such that the first andsecond amino acid sequences can have a common structural domain and/orcommon functional activity. For example, amino acid sequences thatcontain a common structural domain having at least about 60%, or 65%identity, likely 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity, as calculated by a global alignment,to SEQ ID NO:2 are termed substantially identical.

[0088] In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 60%, or 65%identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity, as calculated by a global alignment,to SEQ ID NO:1 or 3 are termed substantially identical.

[0089] “Misexpression or aberrant expression”, as used herein, refers toa non-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

[0090] “Subject”, as used herein, can refer to a mammal, e.g., a human,or to an experimental or animal or disease model. The subject can alsobe a non-human animal, e.g., a horse, cow, goat, or other domesticanimal.

[0091] A “purified preparation of cells”, as used herein, refers to, inthe case of plant or animal cells, an in vitro preparation of cells andnot an entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

[0092] Various aspects of the invention are described in further detailbelow.

[0093] Isolated Nucleic Acid Molecules

[0094] In one aspect, the invention provides, an isolated or purified,nucleic acid molecule that encodes a 14815 polypeptide described herein,e.g., a full length 14815 protein or a fragment thereof, e.g., abiologically active portion of 14815 protein. Also included is a nucleicacid fragment suitable for use as a hybridization probe, which can beused, e.g., to identify a nucleic acid molecule encoding a polypeptideof the invention, 14815 mRNA, and fragments suitable for use as primers,e.g., PCR primers for the amplification or mutation of nucleic acidmolecules.

[0095] In one embodiment, an isolated nucleic acid molecule of theinvention includes the nucleotide sequence shown in SEQ ID NO:1, or aportion of any of this nucleotide sequence. In one embodiment, thenucleic acid molecule includes sequences encoding the human 14815protein (i.e., “the coding region” of SEQ ID NO:1, as shown in SEQ IDNO:3), as well as 5′ untranslated sequences (nucleotides 1 to 182 of SEQID NO:1) and 3′ untranslated sequences (nucleotides 3531 to 3919 of SEQID NO:1). Alternatively, the nucleic acid molecule can include only thecoding region of SEQ ID NO:1 (e.g., SEQ ID NO:3) and, e.g., no flankingsequences which normally accompany the subject sequence. In anotherembodiment, the nucleic acid molecule encodes a sequence correspondingto a fragment of the protein from about amino acid 519 to 779 of SEQ IDNO:2, or a fragment thereof, e.g. about amino acid residues 519 to 600,601 to 700, or 701 to 779 of SEQ ID NO:2.

[0096] In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3, or a portion ofany of these nucleotide sequences. In other embodiments, the nucleicacid molecule of the invention is sufficiently complementary to thenucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3 such that it canhybridize to the nucleotide sequence shown in SEQ ID NO:1 or 3, therebyforming a stable duplex.

[0097] In one embodiment, an isolated nucleic acid molecule of thepresent invention includes a nucleotide sequence which is at leastabout: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more homologous to the entire length of the nucleotidesequence shown in SEQ ID NO: I or SEQ ID NO:3, or a portion, preferablyof the same length, of any of these nucleotide sequences.

[0098] 14815 Nucleic Acid Fragments

[0099] A nucleic acid molecule of the invention can include only aportion of the nucleic acid sequence of SEQ ID NO:1 or 3. For example,such a nucleic acid molecule can include a fragment which can be used asa probe or primer or a fragment encoding a portion of a 14815 protein,e.g., an immunogenic or biologically active portion of a 14815 protein.A fragment can comprise those nucleotides of SEQ ID NO:1, which encode aprotein kinase domain of human 14815. The nucleotide sequence determinedfrom the cloning of the 14815 gene allows for the generation of probesand primers designed for use in identifying and/or cloning other 14815family members, or fragments thereof, as well as 14815 homologs, orfragments thereof, from other species.

[0100] In another embodiment, a nucleic acid includes a nucleotidesequence that includes part, or all, of the coding region and extendsinto either (or both) the 5′ or 3′ noncoding region. Other embodimentsinclude a fragment which includes a nucleotide sequence encoding anamino acid fragment described herein. Nucleic acid fragments can encodea specific domain or site described herein or fragments thereof,particularly fragments thereof which are at least 260 amino acids inlength. Fragments also include nucleic acid sequences corresponding tospecific amino acid sequences described above or fragments thereof.Nucleic acid fragments should not to be construed as encompassing thosefragments that may have been disclosed prior to the invention.

[0101] A nucleic acid fragment can include a sequence corresponding to adomain, region, or functional site described herein. A nucleic acidfragment can also include one or more domain, region, or functional sitedescribed herein. Thus, for example, a 14815 nucleic acid fragment caninclude a sequence corresponding to a protein kinase domain, asdescribed herein.

[0102] 14815 probes and primers are provided. Typically a probe/primeris an isolated or purified oligonucleotide. The oligonucleotidetypically includes a region of nucleotide sequence that hybridizes understringent conditions to at least about 7, 12 or 15, preferably about 20or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense or antisense sequence of SEQ ID NO:1or SEQ ID NO:3, or of a naturally occurring allelic variant or mutant ofSEQ ID NO:1 or SEQ ID NO:3.

[0103] In a preferred embodiment the nucleic acid is a probe which is atleast 5 or 10, and less than 200, more preferably less than 100, or lessthan 50, base pairs in length. It should be identical, or differ by 1,or less than in 5 or 10 bases, from a sequence disclosed herein. Ifalignment is needed for this comparison the sequences should be alignedfor maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.

[0104] A probe or primer can be derived from the sense or anti-sensestrand of a nucleic acid which encodes: a protein kinase domain at aboutamino acid residues 519 to 779 of SEQ ID NO:2, or anarmadillo/beta-catenin-like repeat domain at about amino acid residues198 to 238, 239 to 279, 280 to 320, and 401 to 448 of SEQ ID NO:2. Forexample, a 14815 probe or primer can comprise SEQ ID NO:10, SEQ ID NO:11or SEQ ID NO:12.

[0105] In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a 14815 sequence, e.g., a domain, region, site or othersequence described herein. The primers should be at least 5, 10, or 50base pairs in length and less than 100, or less than 200, base pairs inlength. The primers should be identical, or differ by one base from asequence disclosed herein or from a naturally occurring variant. Forexample, primers suitable for amplifying all or a portion of any of thefollowing regions are provided: a protein kinase domain from about aminoacid 519 to 779 of SEQ ID NO:2, or an armadillo/beta-catenin-like repeatdomain at about amino acid residues 198 to 238, 239 to 279, 280 to 320,and 401 to 448 of SEQ ID NO:2.

[0106] A nucleic acid fragment can encode an epitope bearing region of apolypeptide described herein. Such a fragment, e.g. an antigenicfragment, can comprise a biologically active portion of an 14815polypeptide, as described below, or a subportion thereof, e.g. at least8, 9, 10, 11, 12, 13, 15, 20, 30 or more amino acid residues of SEQ IDNO:2. Thus, the fragment can comprise at least 24, 27, 30, 33, 36, 39,45, 60, 90, or more bases. Such fragments are described in more detailin the “Anti-14815 Antibodies” section below.

[0107] A nucleic acid fragment encoding a “biologically active portionof a 14815 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:1 or 3, which encodes a polypeptidehaving a 14815 biological activity (e.g., the biological activities ofthe 14815 proteins are described herein), expressing the encoded portionof the 14815 protein (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the 14815 protein. Forexample, a nucleic acid fragment encoding a biologically active portionof 14815 includes a protein kinase domain, e.g., amino acid residuesabout 519 to 779 of SEQ ID NO:2, a coiled coil structure, e.g., aminoacid residues about 472 to 500 of SEQ ID NO:2, or anarmadillo/beta-catenin-like repeat domain at about amino acid residues198 to 238, 239 to 279, 280 to 320, and 401 to 448 of SEQ ID NO:2. Anucleic acid fragment encoding a biologically active portion of a 14815polypeptide, can comprise a nucleotide sequence which is greater than69, 72, 84, 87, 120, 123, 780 or more nucleotides in length. Such afragment can encode a polypeptide which at least can bind a nucleotide,e.g. ATP or GTP, or another protein, e.g. another 14815 molecule, adifferent kinase, a cyclin, an ankyrin repeat-containing protein,nucleoplasmin, importin, a ras protein, or vinculin, or a protein orpolypeptide with a tyrosine residue.

[0108] In preferred embodiments, a nucleic acid includes a nucleotidesequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300,2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500,3600, 3700, 3800, 3900 or more nucleotides in length and hybridizesunder stringent hybridization conditions to a nucleic acid molecule ofSEQ ID NO:1 or SEQ ID NO:3.

[0109] 14815 Nucleic Acid Variants

[0110] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.Such differences can be due to degeneracy of the genetic code (andresult in a nucleic acid which encodes the same 14815 proteins as thoseencoded by the nucleotide sequence disclosed herein. In anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a protein having an amino acid sequencewhich differs, by at least 1, but less than 5, 10, 20, 50, or 100 aminoacid residues that shown in SEQ ID NO:2. If alignment is needed for thiscomparison the sequences should be aligned for maximum homology.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences.

[0111] Nucleic acids of the inventor can be chosen for having codons,which are preferred, or non-preferred, for a particular expressionsystem. E.g., the nucleic acid can be one in which at least one codon,at preferably at least 10%, or 20% of the codons has been altered suchthat the sequence is optimized for expression in E. coli, yeast, human,insect, or CHO cells.

[0112] Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

[0113] In a preferred embodiment, the nucleic acid differs from that ofSEQ ID NO:1 or 3, e.g., as follows: by at least one but less than 10,20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20%of the nucleotides in the subject nucleic acid. If necessary for thisanalysis the sequences should be aligned for maximum homology. “Looped”out sequences from deletions or insertions, or mismatches, areconsidered differences.

[0114] Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%, at least about 55%, typically atleast about 70-75%, more typically at least about 80-85%, and mosttypically at least about 90-95% or more identical to the nucleotidesequence shown in SEQ ID NO:2 or a fragment of this sequence. Suchnucleic acid molecules can readily be identified as being able tohybridize under stringent conditions, to the nucleotide sequence shownin SEQ ID NO 2 or a fragment of the sequence. Nucleic acid moleculescorresponding to orthologs, homologs, and allelic variants of the 14815cDNAs of the invention can further be isolated by mapping to the samechromosome or locus as the 14815 gene. For example, the 14815 gene canbe found on human chromosome 14.

[0115] Preferred variants include those that are correlated withphosphorylation of a protein, e.g. phosphorylation of a tyrosine residueon a protein, the binding of a nucleotide, e.g. ATP or GTP, or thebinding of another protein, e.g. another 14815 molecule, a differentkinase, a cyclin, an ankyrin repeat-containing protein, nucleoplasmin,importin, a ras protein, or vinculin, or a protein or polypeptide with atyrosine residue.

[0116] Allelic variants of 14815, e.g., human 14815, include bothfunctional and non-functional proteins. Functional allelic variants arenaturally occurring amino acid sequence variants of the 14815 proteinwithin a population that maintain the ability to bind a molecule, e.g.,a nucleotide (e.g. adenosine triphosphate or guanine triphosphate) andcatalyze the transfer of a functional group, e.g. a phosphate, from thenucleotide to a protein, e.g. to a tyrosine, serine or threonine residueon the protein; the ability to modulate the cell cycle or cell death.Functional allelic variants will typically contain only conservativesubstitution of one or more amino acids of SEQ ID NO:2, or substitution,deletion or insertion of non-critical residues in non-critical regionsof the protein. Non-functional allelic variants are naturally-occurringamino acid sequence variants of the 14815, e.g., human 14815, proteinwithin a population that do not have the ability to bind a molecule,e.g., a nucleotide (e.g. adenosine triphosphate or guanine triphosphate)and catalyze the transfer of a functional group, e.g. a phosphate, fromthe nucleotide to a protein, e.g. to a tyrosine, serine or threonineresidue on the protein; the ability to modulate the cell cycle or celldeath. Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion, or prematuretruncation of the amino acid sequence of SEQ ID NO:2, or a substitution,insertion, or deletion in critical residues or critical regions of theprotein. For example, changes can be made in the protein kinase domain,in one, two, three, or four armadillo/beta-catenin-like repeat domainsof 14815 protein kinase. As an example of a change in the protein kinasedomain, nucleotides in the codon encoding K-548 of SEQ ID NO:2 can bechanged, e.g. changes in nucleotides 1824 and 1825 of SEQ ID NO:1 ornucleotides 1642 or 1643 of SEQ ID NO:3 can inhibit a biologicalactivity, e.g. protein phosphorylation activity, e.g. nucleotide (e.g.ATP or GTP) binding activity, of a 14815 polypeptide. As anotherexample, nucleotides in the codon encoding D-655 of SEQ ID NO:2 can bechanged, e.g. changes in nucleotides 2146 and 2147 of SEQ ID NO:1 ornucleotides 1963 and 1964 of SEQ ID NO:3 can inhibit a biologicalactivity, e.g. protein phosphorylation activity, of a 14815 polypeptide.In another example, nucleotides in regions of SEQ ID NO:1 or SEQ ID NO:3encoding the coiled coil structure, e.g. nucleotides encoding amino acidresidues in regions amino acids residues 472 to 500 of SEQ ID NO:2 canbe changed to alter oligomerization or protein binding of a 14815protein. In further examples, nucleotides in regions of SEQ ID NO:1 orSEQ ID NO:3 encoding one or more of the armadillo/beta-catenin-likerepeat domains, e.g. nucleotides encoding amino acid residues in regionsof amino acids 198 to 238, 239 to 279, 280 to 320, and 401 to 448 of SEQID NO:2 can be changed to alter protein binding, e.g. binding to anotherprotein, e.g. another 14815 molecule, a different kinase, a cyclin, anankyrin repeat-containing protein, nucleoplasmin, importin, a rasprotein, or vinculin, or a protein or polypeptide with a tyrosineresidue of a 14815 polypeptide. Following mutagenesis, the encodedprotein can be expressed recombinantly, and the activity of the proteincan be determined using standard assay techniques.

[0117] Moreover, nucleic acid molecules encoding other 14815 familymembers and, thus, which have a nucleotide sequence which differs fromthe 14815 sequences of SEQ ID NO:1 or SEQ ID NO:3 are intended to bewithin the scope of the invention.

[0118] Antisense Nucleic Acid Molecules, Ribozymes and Modified 14815Nucleic Acid Molecules

[0119] In another aspect, the invention features, an isolated nucleicacid molecule which is antisense to 14815. An “antisense” nucleic acidcan include a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. The antisense nucleic acid can be complementary to an entire14815 coding strand, or to only a portion thereof (e.g., the codingregion of human 14815 corresponding to SEQ ID NO:3). In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding 14815 (e.g., the 5′ and 3′ untranslated regions).

[0120] An antisense nucleic acid can be designed such that it iscomplementary to the entire coding region of 14815 mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of 14815 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of 14815 mRNA, e.g., between the −10 and +10regions of the target gene nucleotide sequence of interest. An antisenseoligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[0121] An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. The antisense nucleicacid also can be produced biologically using an expression vector intowhich a nucleic acid has been subcloned in an antisense orientation(i.e., RNA transcribed from the inserted nucleic acid will be of anantisense orientation to a target nucleic acid of interest, describedfurther in the following subsection).

[0122] The antisense nucleic acid molecules of the invention aretypically administered to a subject (e.g., by direct injection at atissue site), or generated in situ such that they hybridize with or bindto cellular mRNA and/or genomic DNA encoding a 14815 protein to therebyinhibit expression of the protein, e.g., by inhibiting transcriptionand/or translation. Alternatively, antisense nucleic acid molecules canbe modified to target selected cells and then administered systemically.For systemic administration, antisense molecules can be modified suchthat they specifically or selectively bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

[0123] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

[0124] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. A ribozyme having specificity for a14815-encoding nucleic acid can include one or more sequencescomplementary to the nucleotide sequence of a 14815 cDNA disclosedherein (i.e., SEQ ID NO:1 or SEQ ID NO:3), and a sequence having knowncatalytic sequence responsible for mRNA cleavage (see U.S. Pat. No.5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a 14815-encoding mRNA. See,e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, 14815 mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.

[0125] 14815 gene expression can be inhibited by targeting nucleotidesequences complementary to the regulatory region of the 14815 (e.g., the14815 promoter and/or enhancers) to form triple helical structures thatprevent transcription of the 14815 gene in target cells. See generally,Helene (1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15. Thepotential sequences that can be targeted for triple helix formation canbe increased by creating a so-called “switchback” nucleic acid molecule.Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′manner, such that they base pair with first one strand of a duplex andthen the other, eliminating the necessity for a sizeable stretch ofeither purines or pyrimidines to be present on one strand of a duplex.

[0126] The invention also provides detectably labeled oligonucleotideprimer and probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

[0127] A 14815 nucleic acid molecule can be modified at the base moiety,sugar moiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup et al. (1996)Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms“peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., aDNA mimic, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of a PNA can allow for specifichybridization to DNA and RNA under conditions of low ionic strength. Thesynthesis of PNA oligomers can be performed using standard solid phasepeptide synthesis protocols as described in Hyrup et al. (1996) supra;Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. 93: 14670-675.

[0128] PNAs of 14815 nucleic acid molecules can be used in therapeuticand diagnostic applications. For example, PNAs can be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, for example, inducing transcription or translation arrest orinhibiting replication. PNAs of 14815 nucleic acid molecules can also beused in the analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., S1 nucleases (Hyrup etal. (1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup et al. (1996) supra; Perry-O'Keefe supra).

[0129] In other embodiments, the oligonucleotide can include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier(see, e.g., PCT Publication No. W089/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents. (see, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotide can be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

[0130] The invention also includes molecular beacon oligonucleotideprimer and probe molecules having at least one region which iscomplementary to a 14815 nucleic acid of the invention, twocomplementary regions one having a fluorophore and one a quencher suchthat the molecular beacon is useful for quantitating the presence of the14815 nucleic acid of the invention in a sample. Molecular beaconnucleic acids are described, for example, in Lizardi et al., U.S. Pat.No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak etal., U.S. Pat. No. 5,876,930.

[0131] Isolated 14815 Polypeptides

[0132] In another aspect, the invention features, an isolated 14815protein, or fragment, e.g., a biologically active portion, for use,e.g., in screening assays, as therapeutic or diagnostic targets, or asimmunogens or antigens to raise or test (or more generally to bind)anti-14815 antibodies. 14815 protein can be isolated from cells ortissue sources using standard protein purification techniques. 14815protein or fragments thereof can be produced by recombinant DNAtechniques or synthesized chemically.

[0133] Polypeptides of the invention include those which arise as aresult of the existence of multiple genes, alternative transcriptionevents, alternative RNA splicing events, and alternative translationaland post-translational events. The polypeptide can be expressed insystems, e.g., cultured cells, which result in substantially the samepost-translational modifications present when the polypeptide isexpressed in a native cell, or in systems which result in the alterationor omission of post-translational modifications, e.g., glycosylation orcleavage, present in a native cell.

[0134] In a preferred embodiment, a 14815 polypeptide has one or more ofthe following characteristics:

[0135] it has the ability to bind a molecule, e.g., a nucleotide (e.g.adenosine triphosphate or guanine triphosphate);

[0136] the ability to bind a protein substrate, e.g. a tyrosine, serineor threonine-containing protein;

[0137] the ability to catalyze the transfer of a functional group, e.g.a phosphate, from the nucleotide to the protein, e.g. to a tyrosine,serine or threonine residue on the protein;

[0138] the ability to bind a second protein, e.g. another 14815molecule, a different kinase, a cyclin, an ankyrin repeat-containingprotein, nucleoplasmin, importin, a ras protein, or vinculin;

[0139] the ability to regulate transmission of signals from cellularreceptors, e.g., cell growth factor receptors;

[0140] the ability to modulate the entry of cells, e.g., precursorcells, into the cell cycle, e.g. mitosis or meiosis;

[0141] the ability to modulate cell death, e.g. apoptosis;

[0142] it has a molecular weight, e.g., a deduced molecular weight,preferably ignoring any contribution of post translationalmodifications, amino acid composition or other physical characteristicof a 14815 polypeptide, e.g., a polypeptide of SEQ ID NO:2;

[0143] it has an overall sequence identity of at least 60%, preferablyat least 70%, more preferably at least 80, 90, or 95%, as calculated bya global alignment, with a polypeptide of SEQ ID NO:2;

[0144] it can be found in breast tissue and primary breast tumors, butmuch less in metastases from the primary tumor;

[0145] it has a protein kinase domain which is preferably about 70%,80%, 90% or 95% identical to amino acid residues about 519 to 779 of SEQID NO:2;

[0146] it has at least one, two, three, preferably fourarmadillo/beta-catenin-like repeat domains which are preferably about70%, 80%, 90% or 95% identical to amino acid residues about 198 to 238,239 to 279, 280 to 320, and 401 to 448 of SEQ ID NO:2;

[0147] In a preferred embodiment, the 14815 protein, or fragmentthereof, differs from the corresponding sequence in SEQ [D NO:2. In oneembodiment it differs by at least one but by less than 15, 10 or 5 aminoacid residues. In another it differs from the corresponding sequence inSEQ ID NO:2 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ IDNO:2. (If this comparison requires alignment the sequences should bealigned for maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.) The differencesare, preferably, differences or changes at a non-essential residue or aconservative substitution. In a preferred embodiment, the differencesare not in the protein kinase domain at about residues 519 to 779 of SEQID NO:2 or in one of the armadillo/beta-catenin-like repeat domains atabout residues 198 to 238, 239 to 279, 280 to 320, and 401 to 448 of SEQID NO:2. In another embodiment one or more differences are in theprotein kinase domain at about residues 519 to 779 of SEQ ID NO:2 or inone of the armadillo/beta-catenin-like repeat domains at about residues198 to 238, 239 to 279, 280 to 320, and 401 to 448 of SEQ ID NO:2.

[0148] Other embodiments include a protein that contains one or morechanges in amino acid sequence, e.g., a change in an amino acid residuewhich is not essential for activity. Such 14815 proteins differ in aminoacid sequence from SEQ ID NO:2, yet retain biological activity.

[0149] In one embodiment, the protein includes an amino acid sequence atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or morehomologous to SEQ ID NO:2 as calculated by a global alignment. Inanother embodiment, the protein includes fragments or regions homologousto fragments, at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% ormore homologous to a fragment of SEQ ID NO:2, as calculated by a gappedalignment. A fragment of a 14815 protein can be a domain, e.g. a proteinkinase domain at about residues 519 to 779 of SEQ ID NO:2 or a fragmentthereof, e.g. about amino acid residues 519 to 600, 601 to 700, or 701to 779 of SEQ ID NO:2, a coiled coil structure, e.g. about amino acidresidues 472 to 500 of SEQ ID NO:2, or an armadillo/beta-catenin-likerepeat domain, e.g. at about residues 198 to 238, 239 to 279, 280 to320, and 401 to 448 of SEQ ID NO:2.

[0150] A 14815 protein or fragment is provided which varies from thesequence of SEQ ID NO:2 in regions defined by amino acids about 1 to 519or 780 to 1115 by at least one but by less than 15, 10 or 5 amino acidresidues in the protein or fragment but which does not differ from SEQID NO:2 in regions defined by amino acids about 519 to 779. (If thiscomparison requires alignment the sequences should be aligned formaximum homology. “Looped” out sequences from deletions or insertions,or mismatches, are considered differences.) In some embodiments thedifference is at a non-essential residue or is a conservativesubstitution, while in others the difference is at an essential residueor is a non-conservative substitution.

[0151] In one embodiment, a biologically active portion of a 14815protein includes a protein kinase domain. In other embodiments,biologically active portions of a 14815 protein include at least onearmadillo/beta-catenin-like repeat domain or a coiled coil structure.Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native 14815protein.

[0152] In a preferred embodiment, the 14815 protein has an amino acidsequence shown in SEQ ID NO:2. In other embodiments, the 14815 proteinis sufficiently or substantially identical to SEQ ID NO:2. In yetanother embodiment, the 14815 protein is sufficiently or substantiallyidentical to SEQ ID NO:2 and retains the functional activity of theprotein of SEQ ID NO:2, as described in detail in the subsections above.

[0153] 14815 Chimeric or Fusion Proteins

[0154] In another aspect, the invention provides 14815 chimeric orfusion proteins. As used herein, a 14815 ”chimeric protein” or “fusionprotein” includes a 14815 polypeptide linked to a non-14815 polypeptide.A “non-14815 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein which is not substantiallyhomologous to the 14815 protein, e.g., a protein which is different fromthe 14815 protein and which is derived from the same or a differentorganism. The 14815 polypeptide of the fusion protein can correspond toall or a portion e.g., a fragment described herein of a 14815 amino acidsequence, e.g. a domain, e.g. a protein kinase domain or a fragmentthereof, e.g. about amino acid residues 519 to 779, 519 to 600, 601 to700, or 701 to 779 of SEQ ID NO:2, a coiled coil structure, e.g. aboutamino acid residues 472 to 500 of SEQ ID NO:2, or anarmadillo/beta-catenin-like repeat domain, e.g. at about residues 198 to238, 239 to 279, 280 to 320, and 401 to 448 of SEQ ID NO:2. In apreferred embodiment, a 14815 fusion protein includes at least one (ortwo) biologically active portion of a 14815 protein. The non-14815polypeptide can be fused to the N-terminus or C-terminus of the 14815polypeptide.

[0155] The fusion protein can include a moiety which has a high affinityfor a ligand. For example, the fusion protein can be a GST-14815 fusionprotein in which the 14815 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant 14815. Alternatively, the fusion protein can be a 14815protein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of 14815 can be increased through use of a heterologous signalsequence.

[0156] Fusion proteins can include all or a part of a serum protein,e.g., a portion of an immunoglobulin (e.g., IgG, IgA, or IgE), e.g., anFe region and/or the hinge C1 and C2 sequences of an immunoglobulin orhuman serum albumin.

[0157] The 14815 fusion proteins of the invention can be incorporatedinto pharmaceutical compositions and administered to a subject in vivo.The 14815 fusion proteins can be used to affect the bioavailability of a14815 substrate. 14815 fusion proteins can be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a 14815 protein; (ii)mis-regulation of the 14815 gene; and (iii) aberrant post-translationalmodification of a 14815 protein.

[0158] Moreover, the 14815-fusion proteins of the invention can be usedas immunogens to produce anti-14815 antibodies in a subject, to purify14815 ligands and in screening assays to identify molecules whichinhibit the interaction of 14815 with a 14815 substrate.

[0159] Expression vectors are commercially available that already encodea fusion moiety (e.g., a GST polypeptide). A 14815-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the 14815 protein. Variants of 14815 Proteins

[0160] In another aspect, the invention also features a variant of a14815 polypeptide, e.g., which functions as an agonist (mimetics) or asan antagonist. Variants of the 14815 proteins can be generated bymutagenesis, e.g., discrete point mutation, the insertion or deletion ofsequences or the truncation of a 14815 protein. An agonist of the 14815proteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a 14815protein. An antagonist of a 14815 protein can inhibit one or more of theactivities of the naturally occurring form of the 14815 protein by, forexample, competitively modulating a 14815-mediated activity of a 14815protein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. Preferably, treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of the14815 protein.

[0161] Variants of a 14815 protein can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of a 14815protein for agonist or antagonist activity.

[0162] Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a 14815 protein coding sequence can be used to generate avariegated population of fragments for screening and subsequentselection of variants of a 14815 protein.

[0163] Variants in which a cysteine residues is added or deleted or inwhich a residue which is glycosylated is added or deleted areparticularly preferred.

[0164] Other variants can alter, eliminate or inhibit the biologicalactivity (e.g. the protein kinase activity) of the 14815 polypeptide setforth in SEQ ID NO:2. For example, a variant of a 14815 polypeptide canhave an amino acid other than K at position 548 or a variant can have anamino acid other than D at position 655 of SEQ ID NO:2 such that theresulting polypeptide does not have protein kinase activity.

[0165] Methods for screening gene products of combinatorial librariesmade by point mutations or truncation, and for screening cDNA librariesfor gene products having a selected property are known in the art.Recursive ensemble mutagenesis (REM), a new technique which enhances thefrequency of functional mutants in the libraries, can be used incombination with the screening assays to identify 14815 variants (Arkinand Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave etal. (1993) Protein Engineering 6:327-331).

[0166] Cell based assays can be exploited to analyze a variegated 14815library. For example, a library of expression vectors can be transfectedinto a cell line, e.g., a cell line, which ordinarily responds to 14815in a substrate-dependent manner. The transfected cells are thencontacted with 14815 and the effect of the expression of the mutant onsignaling by the 14815 substrate can be detected, e.g., by measuringphosphorylation of a protein, e.g. phosphorylation of a tyrosine residueon a protein, the binding of a nucleotide, e.g. ATP or GTP, to a 14815polypeptide, or the binding of a 14815 polypeptide to another protein,e.g. another 14815 protein, a different kinase, a cyclin, an ankyrinrepeat-containing protein, nucleoplasmin, importin, a ras protein, orvinculin. Plasmid DNA can then be recovered from the cells which scorefor inhibition, or alternatively, potentiation of signaling by the 14815substrate, and the individual clones further characterized.

[0167] In another aspect, the invention features a method of making a14815 polypeptide, e.g., a peptide having a non-wild type activity,e.g., an antagonist, agonist, or super agonist of a naturally occurring14815 polypeptide, e.g., a naturally occurring 14815 polypeptide. Themethod includes altering the sequence of a 14815 polypeptide, e.g.,altering the sequence, e.g., by substitution or deletion of one or moreresidues of a non-conserved region, a domain or residue disclosedherein, and testing the altered polypeptide for the desired activity.

[0168] In another aspect, the invention features a method of making afragment or analog of a 14815 polypeptide a biological activity of anaturally occurring 14815 polypeptide. The method includes altering thesequence, e.g., by substitution or deletion of one or more residues, ofa 14815 polypeptide, e.g., altering the sequence of a non-conservedregion, or a domain or residue described herein, and testing the alteredpolypeptide for the desired activity.

[0169] Anti-14815 Antibodies

[0170] In another aspect, the invention provides an anti-14815 antibody.The term “antibody” as used herein refers to an immunoglobulin moleculeor immunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include scFV and dcFV fragments, Fab and F(ab′)₂ fragmentswhich can be generated by treating the antibody with an enzyme such aspapain or pepsin, respectively.

[0171] The antibody can be a polyclonal, monoclonal, recombinant, e.g.,a chimeric or humanized, fully human, non-human, e.g., murine, or singlechain antibody. In a preferred embodiment it has effector function andcan fix complement. The antibody can be coupled to a toxin or imagingagent.

[0172] A full-length 14815 protein or, antigenic peptide fragment of14815 can be used as an immunogen or can be used to identify anti-14815antibodies made with other immunogens, e.g., cells, membranepreparations, and the like. The antigenic peptide of 14815 shouldinclude at least 8 amino acid residues of the amino acid sequence shownin SEQ ID NO:2 and encompasses an epitope of 14815. Preferably, theantigenic peptide includes at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

[0173] Fragments of 14815 which include residues about 51 to 61, fromabout 90 to 104, and from about 802 to 815 of SEQ ID NO:2 can be used tomake, e.g., used as immunogens or used to characterize the specificityof an antibody, antibodies against hydrophilic regions of the 14815protein (see FIG. 1). Similarly, fragments of 14815 which includeresidues about 135 to 143, from about 225 to 237, and from about 634 to642 of SEQ ID NO:2 can be used to make an antibody against a hydrophobicregion of the 14815 protein; fragments of 14815 which include residuesabout 1 to 50, about 105 to 197, about 321 to 400, about 450 to 515,about 780 to 900, about 901 to 1000 or about 1001 to 1115 of SEQ ID NO:2can be used to make an antibody against an intracellular region of the14815 protein; a fragment of 14815 which includes residues about 519 to600, about 601 to 700, or about 701 to 779 of SEQ ID NO:2 can be used tomake an antibody against the protein kinase region of the 14815 protein;a fragment of 14815 which includes about residues 198 to 238, about 239to 279, about 280 to 320, and about 401 to 448 of SEQ ID NO:2 can beused to make an antibody against an armadillo/beta-catenin-like repeatdomain of the 14815 protein.

[0174] Antibodies reactive with, or specific or selective for, any ofthese regions, or other regions or domains described herein areprovided.

[0175] Preferred epitopes encompassed by the antigenic peptide areregions of 14815 located on the surface of the protein, e.g.,hydrophilic regions, as well as regions with high antigenicity. Forexample, an Emini surface probability analysis of the human 14815protein sequence can be used to indicate the regions that have aparticularly high probability of being localized to the surface of the14815 protein and are thus likely to constitute surface residues usefulfor targeting antibody production.

[0176] In a preferred embodiment the antibody binds an epitope on anydomain or region on 14815 proteins described herein.

[0177] Additionally, chimeric, humanized, and completely humanantibodies are also within the scope of the invention. Chimeric,humanized, but most preferably, completely human antibodies aredesirable for applications which include repeated administration, e.g.,therapeutic treatment of human patients, and some diagnosticapplications.

[0178] Chimeric and humanized monoclonal antibodies, comprising bothhuman and non-human portions, can be made using standard recombinant DNAtechniques. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in Robinson et al. International Application No.PCT/US86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al. EuropeanPatent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559).

[0179] A humanized or complementarity determining region (CDR)-graftedantibody will have at least one or two, but generally all threerecipient CDR's (of heavy and or light immuoglobulin chains) replacedwith a donor CDR. The antibody may be replaced with at least a portionof a non-human CDR or only some of the CDR's may be replaced withnon-human CDR's. It is only necessary to replace the number of CDR'srequired for binding of the humanized antibody to a 14815 or a fragmentthereof. Preferably, the donor will be a rodent antibody, e.g., a rat ormouse antibody, and the recipient will be a human framework or a humanconsensus framework. Typically, the immunoglobulin providing the CDR'sis called the “donor” and the immunoglobulin providing the framework iscalled the “acceptor.” In one embodiment, the donor immunoglobulin is anon-human (e.g., rodent). The acceptor framework is anaturally-occurring (e.g., a human) framework or a consensus framework,or a sequence about 85% or higher, preferably 90%, 95%, 99% or higheridentical thereto.

[0180] As used herein, the term “consensus sequence” refers to thesequence formed from the most frequently occurring amino acids (ornucleotides) in a family of related sequences (See e.g., Winnaker,(1987) From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany). Ina family of proteins, each position in the consensus sequence isoccupied by the amino acid occurring most frequently at that position inthe family. If two amino acids occur equally frequently, either can beincluded in the consensus sequence. A “consensus framework” refers tothe framework region in the consensus immunoglobulin sequence.

[0181] An antibody can be humanized by methods known in the art.Humanized antibodies can be generated by replacing sequences of the Fvvariable region which are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison (1985) Science229:1202-1207, by Oi et al. (1986) BioTechniques 4:214, and by Queen etal. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, the contents ofall of which are hereby incorporated by reference. Those methods includeisolating, manipulating, and expressing the nucleic acid sequences thatencode all or part of immunoglobulin Fv variable regions from at leastone of a heavy or light chain. Sources of such nucleic acid are wellknown to those skilled in the art and, for example, may be obtained froma hybridoma producing an antibody against a 14815 polypeptide orfragment thereof. The recombinant DNA encoding the humanized antibody,or fragment thereof, can then be cloned into an appropriate expressionvector.

[0182] Humanized or CDR-grafted antibodies can be produced byCDR-grafting or CDR substitution, wherein one, two, or all CDR's of animmunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539;Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science239:1534; Beidler et al. (1988) J. Immunol. 141:4053-4060; Winter U.S.Pat. No. 5,225,539, the contents of all of which are hereby expresslyincorporated by reference. Winter describes a CDR-grafting method whichmay be used to prepare the humanized antibodies of the present invention(UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S.Pat. No. 5,225,539), the contents of which is expressly incorporated byreference.

[0183] Also within the scope of the invention are humanized antibodiesin which specific amino acids have been substituted, deleted or added.Preferred humanized antibodies have amino acid substitutions in theframework region, such as to improve binding to the antigen. Forexample, a humanized antibody will have framework residues identical tothe donor framework residue or to another amino acid other than therecipient framework residue. To generate such antibodies, a selected,small number of acceptor framework residues of the humanizedimmunoglobulin chain can be replaced by the corresponding donor aminoacids. Preferred locations of the substitutions include amino acidresidues adjacent to the CDR, or which are capable of interacting with aCDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting aminoacids from the donor are described in U.S. Pat. No. 5,585,089, e.g.,columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 ofU.S. Pat. No. 5,585,089, the contents of which are hereby incorporatedby reference. Other techniques for humanizing antibodies are describedin Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[0184] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Such antibodies can be producedusing transgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Fremont, Calif.) and Medarex, Inc. (Princeton, N.J.), canbe engaged to provide human antibodies directed against a selectedantigen using technology similar to that described above.

[0185] Completely human antibodies that recognize a selected epitope canbe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

[0186] The anti-14815 antibody can be a single chain antibody. Asingle-chain antibody (scFV) can be engineered as described in, forexample, Colcher et al. (1999) Ann. N Y Acad. Sci. 880:263-80; andReiter (1996) Clin. Cancer Res. 2:245-52. The single chain antibody canbe dimerized or multimerized to generate multivalent antibodies havingspecificities for different epitopes of the same target 14815 protein.

[0187] In a preferred embodiment, the antibody has reduced or no abilityto bind an Fc receptor. For example, it is an isotype or subtype,fragment or other mutant, which does not support binding to an Fcreceptor, e.g., it has a mutagenized or deleted Fc receptor bindingregion.

[0188] An antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive ion. A cytotoxin or cytotoxic agent includes any agent thatis detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, coichicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g.,maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos.5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof.Therapeutic agents include, but are not limited to, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids). Radioactiveions include, but are not limited to iodine, yttrium and praseodymium.

[0189] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, thetherapeutic moiety may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; aprotein such as tumor necrosis factor, α-interferon, β-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

[0190] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980.

[0191] An anti-14815 antibody (e.g., monoclonal antibody) can be used toisolate 14815 by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, an anti-14815 antibody can be used todetect 14815 protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of theprotein. Anti-14815 antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance (i.e., antibody labelling). Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0192] In preferred embodiments, an antibody can be made by immunizingwith a purified 14815 antigen, or a fragment thereof, e.g., a fragmentdescribed herein, a membrane associated antigen, tissues, e.g., crudetissue preparations, whole cells, preferably living cells, lysed cells,or cell fractions, e.g., cytosolic supernatants, membrane fractions orperoxisomal fractions.

[0193] Antibodies which bind only a native 14815 protein, only denaturedor otherwise non-native 14815 protein, or which bind both, are withinthe invention. Antibodies with linear or conformational epitopes arewithin the invention. Conformational epitopes sometimes can beidentified by identifying antibodies which bind to native but notdenatured 14815 protein.

[0194] Recombinant Expression Vectors, Host Cells and GeneticallyEngineered Cells

[0195] In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

[0196] A vector can include a 14815 nucleic acid in a form suitable forexpression of the nucleic acid in a host cell. Preferably therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid sequence to be expressed. Theterm “regulatory sequence” includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those which direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or polypeptides, including fusion proteins or polypeptides,encoded by nucleic acids as described herein (e.g., 14815 proteins,mutant forms of 14815 proteins, fusion proteins, and the like).

[0197] The recombinant expression vectors of the invention can bedesigned for expression of 14815 proteins in prokaryotic or eukaryoticcells. For example, polypeptides of the invention can be expressed in E.coli, insect cells (e.g., using baculovirus expression vectors), yeastcells or mammalian cells. Suitable host cells are discussed further inGoeddel, (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

[0198] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

[0199] Purified fusion proteins can be used in 14815 activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific or selective for 14815 proteins. In apreferred embodiment, a fusion protein expressed in a retroviralexpression vector of the present invention can be used to infect bonemarrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time has passed (e.g., six weeks).

[0200] To maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman (1990) GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. 119-128). Another strategy is to alter the nucleic acidsequence of the nucleic acid to be inserted into an expression vector sothat the individual codons for each amino acid are those preferentiallyutilized in E. coli (Wada et al., (1992) Nucleic Acids Res.20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

[0201] The 14815 expression vector can be a yeast expression vector, avector for expression in insect cells, e.g., a baculovirus expressionvector or a vector suitable for expression in mammalian cells.

[0202] When used in mammalian cells, the expression vector's controlfunctions are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40.

[0203] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid). Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for example,the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0204] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus. For a discussion of the regulation of gene expressionusing antisense genes see Weintraub et al., (1986) Reviews—Trends inGenetics 1:1.

[0205] Another aspect the invention provides a host cell which includesa nucleic acid molecule described herein, e.g., a 14815 nucleic acidmolecule within a recombinant expression vector or a 14815 nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but also to the progeny orpotential progeny of such a cell. Because certain modifications canoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0206] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a 14815 protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary (CHO) cells or CV-1 origin, SV-40 (COS) cells). Other suitablehost cells are known to those skilled in the art.

[0207] Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

[0208] A host cell of the invention can be used to produce (i.e.,express) a 14815 protein. Accordingly, the invention further providesmethods for producing a 14815 protein using the host cells of theinvention. In one embodiment, the method includes culturing the hostcell of the invention (into which a recombinant expression vectorencoding a 14815 protein has been introduced) in a suitable medium suchthat a 14815 protein is produced. In another embodiment, the methodfurther includes isolating a 14815 protein from the medium or the hostcell.

[0209] In another aspect, the invention features, a cell or purifiedpreparation of cells which include a 14815 transgene, or which otherwisemisexpress 14815. The cell preparation can consist of human or non-humancells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, orpig cells. In preferred embodiments, the cell or cells include a 14815transgene, e.g., a heterologous form of a 14815, e.g., a gene derivedfrom humans (in the case of a non-human cell). The 14815 transgene canbe misexpressed, e.g., overexpressed or underexpressed. In otherpreferred embodiments, the cell or cells include a gene whichmisexpresses an endogenous 14815, e.g., a gene the expression of whichis disrupted, e.g., a knockout. Such cells can serve as a model forstudying disorders which are related to mutated or misexpressed 14815alleles or for use in drug screening.

[0210] In another aspect, the invention features, a human cell, e.g., ahematopoietic stem cell, transformed with nucleic acid which encodes asubject 14815 polypeptide.

[0211] Also provided are cells, preferably human cells, e.g., humanhematopoietic or fibroblast cells, in which an endogenous 14815 is underthe control of a regulatory sequence that does not normally control theexpression of the endogenous 14815 gene. The expression characteristicsof an endogenous gene within a cell, e.g., a cell line or microorganism,can be modified by inserting a heterologous DNA regulatory element intothe genome of the cell such that the inserted regulatory element isoperably linked to the endogenous 14815 gene. For example, an endogenous14815 gene which is “transcriptionally silent,” e.g., not normallyexpressed, or expressed only at very low levels, can be activated byinserting a regulatory element which is capable of promoting theexpression of a normally expressed gene product in that cell. Techniquessuch as targeted homologous recombinations, can be used to insert theheterologous DNA as described in, e.g., Chappel, U.S. Pat. No.5,272,071; WO 91/06667, published in May 16, 1991.

[0212] Transgenic Animals

[0213] The invention provides non-human transgenic animals. Such animalsare useful for studying the function and/or activity of a 14815 proteinand for identifying and/or evaluating modulators of 14815 activity. Asused herein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, and the like. A transgene is exogenous DNA or arearrangement, e.g., a deletion of endogenous chromosomal DNA, whichpreferably is integrated into or occurs in the genome of the cells of atransgenic animal. A transgene can direct the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal, other transgenes, e.g., a knockout, reduce expression. Thus, atransgenic animal can be one in which an endogenous 14815 gene has beenaltered by, e.g., by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

[0214] Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to a transgene of the invention in order to direct expression ofa 14815 protein to particular cells. A transgenic founder animal can beidentified based upon the presence of a 14815 transgene in its genomeand/or expression of 14815 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a 14815 protein can further be bred to othertransgenic animals carrying other transgenes.

[0215] 14815 proteins or polypeptides can be expressed in transgenicanimals or plants, e.g., a nucleic acid encoding the protein orpolypeptide can be introduced into the genome of an animal. In preferredembodiments the nucleic acid is placed under the control of a tissuespecific promoter, e.g., a milk or egg specific promoter, and recoveredfrom the milk or eggs produced by the animal. Suitable animals are mice,pigs, cows, goats, and sheep.

[0216] The invention also includes a population of cells from atransgenic animal, as discussed, e.g., below.

[0217] Uses

[0218] The nucleic acid molecules, proteins, protein homologs, andantibodies described herein can be used in one or more of the followingmethods: a) screening assays; b) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenetics); and c) methods of treatment (e.g., therapeutic andprophylactic).

[0219] The isolated nucleic acid molecules of the invention can be used,for example, to express a 14815 protein (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect a 14815 mRNA (e.g., in a biological sample) or a geneticalteration in a 14815 gene, and to modulate 14815 activity, as describedfurther below. The 14815 proteins can be used to treat disorderscharacterized by insufficient or excessive production of a 14815substrate or production of 14815 inhibitors. In addition, the 14815proteins can be used to screen for naturally occurring 14815 substrates,to screen for drugs or compounds which modulate 14815 activity, as wellas to treat disorders characterized by insufficient or excessiveproduction of 14815 protein or production of 14815 protein forms whichhave decreased, aberrant or unwanted activity compared to 14815 wildtype protein (e.g., aberrant or deficient kinase function orexpression). Moreover, the anti-14815 antibodies of the invention can beused to detect and isolate 14815 proteins, regulate the bioavailabilityof 14815 proteins, and modulate 14815 activity.

[0220] A method of evaluating a compound for the ability to interactwith, e.g., bind, a subject 14815 polypeptide is provided. The methodincludes: contacting the compound with the subject 14815 polypeptide;and evaluating ability of the compound to interact with, e.g., to bindor form a complex with the subject 14815 polypeptide. This method can beperformed in vitro, e.g., in a cell free system, or in vivo, e.g., in atwo-hybrid interaction trap assay. This method can be used to identifynaturally occurring molecules which interact with subject 14815polypeptide. It can also be used to find natural or synthetic inhibitorsof subject 14815 polypeptide. Screening methods are discussed in moredetail below.

[0221] Screening Assays:

[0222] The invention provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., proteins, peptides, peptidomimetics,peptoids, small molecules or other drugs) which bind to 14815 proteins,have a stimulatory or inhibitory effect on, for example, 14815expression or 14815 activity, or have a stimulatory or inhibitory effecton, for example, the expression or activity of a 14815 substrate.Compounds thus identified can be used to modulate the activity of targetgene products (e.g., 14815 genes) in a therapeutic protocol, toelaborate the biological function of the target gene product, or toidentify compounds that disrupt normal target gene interactions.

[0223] In one embodiment, the invention provides assays for screeningcandidate or test compounds which are substrates of a 14815 protein orpolypeptide or a biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to or modulate the activity of a 14815 proteinor polypeptide or a biologically active portion thereof.

[0224] The test compounds of the present invention can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art, including: biological libraries; peptoid libraries (librariesof molecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et al. (1994)J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

[0225] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci.USA 91:11422-426; Zuckermann et al. (1994). J. Med. Chem. 37:2678-85;Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem.Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233-51.

[0226] Libraries of compounds can be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner, U.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

[0227] In one embodiment, an assay is a cell-based assay in which a cellwhich expresses a 14815 protein or biologically active portion thereofis contacted with a test compound, and the ability of the test compoundto modulate 14815 activity is determined. Determining the ability of thetest compound to modulate 14815 activity can be accomplished bymonitoring, for example, phosphorylation of a protein, e.g.phosphorylation of a tyrosine residue on a protein, the binding of anucleotide, e.g. ATP or GTP, to a 14815 polypeptide, or the binding of a14815 polypeptide to another protein, e.g. another 14815 protein, adifferent kinase, a cyclin, an ankyrin repeat-containing protein,nucleoplasmin, importin, a ras protein, or vinculin. The cell, forexample, can be of mammalian origin, e.g., human.

[0228] The ability of the test compound to modulate 14815 binding to acompound, e.g., a 14815 substrate, or to bind to 14815 can also beevaluated. This can be accomplished, for example, by coupling thecompound, e.g., the substrate, with a radioisotope or enzymatic labelsuch that binding of the compound, e.g., the substrate, to 14815 can bedetermined by detecting the labeled compound, e.g., substrate, in acomplex. Alternatively, 14815 could be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulate14815 binding to a 14815 substrate in a complex. For example, compounds(e.g., 14815 substrates) can be labeled with ¹²⁵I, ¹⁴C, ³⁵S or ³H.,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

[0229] The ability of a compound (e.g., a 14815 substrate) to interactwith 14815 with or without the labeling of any of the interactants canbe evaluated. For example, a microphysiometer can be used to detect theinteraction of a compound with 14815 without the labeling of either thecompound or the 14815. McConnell et al. (1992) Science 257:1906-1912. Asused herein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and 14815.

[0230] In yet another embodiment, a cell-free assay is provided in whicha 14815 protein or biologically active portion thereof is contacted witha test compound and the ability of the test compound to bind to the14815 protein or biologically active portion thereof is evaluated.Preferred biologically active portions of the 14815 proteins to be usedin assays of the present invention include fragments which participatein interactions with non-14815 molecules, e.g., fragments with highsurface probability scores.

[0231] Soluble and/or membrane-bound forms of isolated proteins (e.g.,14815 proteins or biologically active portions thereof) can be used inthe cell-free assays of the invention. When membrane-bound forms of theprotein are used, it may be desirable to utilize a solubilizing agent.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl═N,N-dimethyl-3-ammonio-1-propane sulfonate.

[0232] Cell-free assays involve preparing a reaction mixture of thetarget gene protein and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex that can be removed and/or detected.

[0233] The interaction between two molecules can also be detected, e.g.,using fluorescence energy transfer (FET) (see, for example, Lakowicz etal., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule can simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label can be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

[0234] In another embodiment, determining the ability of the 14815protein to bind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander andUrbaniczky (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr.Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA”detects biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore). Changes in the mass at the bindingsurface (indicative of a binding event) result in alterations of therefractive index of light near the surface (the optical phenomenon ofsurface plasmon resonance (SPR)), resulting in a detectable signal whichcan be used as an indication of real-time reactions between biologicalmolecules.

[0235] In one embodiment, the target gene product or the test substanceis anchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

[0236] It may be desirable to immobilize either 14815, an anti-14815antibody or its target molecule to facilitate separation of complexedfrom uncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to a14815 protein, or interaction of a 14815 protein with a target moleculein the presence and absence of a candidate compound, can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/14815 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or 14815 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of 14815binding or activity determined using standard techniques.

[0237] Other techniques for immobilizing either a 14815 protein or atarget molecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated 14815 protein or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

[0238] In order to conduct the assay, the non-immobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously non-immobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific or selective for the immobilizedcomponent (the antibody, in turn, can be directly labeled or indirectlylabeled with, e.g., a labeled anti-Ig antibody).

[0239] In one embodiment, this assay is performed utilizing antibodiesreactive with 14815 protein or target molecules but which do notinterfere with binding of the 14815 protein to its target molecule. Suchantibodies can be derivatized to the wells of the plate, and unboundtarget or 14815 protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the 14815 protein or targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the 14815 protein or target molecule.

[0240] Alternatively, cell free assays can be conducted in a liquidphase. In such an assay, the reaction products are separated fromunreacted components, by any of a number of standard techniques,including but not limited to: differential centrifugation (see, forexample, Rivas and Minton (1993) Trends Biochem Sci 18:284-7);chromatography (gel filtration chromatography, ion-exchangechromatography); electrophoresis (see, e.g., Ausubel et al., eds. (1999)Current Protocols in Molecular Biology, J. Wiley, New York.); andimmunoprecipitation (see, for example, Ausubel et al., eds. (1999)Current Protocols in Molecular Biology, J. Wiley, New York). Such resinsand chromatographic techniques are known to one skilled in the art (see,e.g., Heegaard (1998) J Mol Recognit 11:141-8; Hage and Tweed (1997) JChromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energytransfer can also be conveniently utilized, as described herein, todetect binding without further purification of the complex fromsolution.

[0241] In a preferred embodiment, the assay includes contacting the14815 protein or biologically active portion thereof with a knowncompound which binds 14815 to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a 14815 protein, wherein determining theability of the test compound to interact with a 14815 protein includesdetermining the ability of the test compound to preferentially bind to14815 or biologically active portion thereof, or to modulate theactivity of a target molecule, as compared to the known compound.

[0242] The target gene products of the invention can, in vivo, interactwith one or more cellular or extracellular macromolecules, such asproteins. For the purposes of this discussion, such cellular andextracellular macromolecules are referred to herein as “bindingpartners.” Compounds that disrupt such interactions can be useful inregulating the activity of the target gene product. Such compounds caninclude, but are not limited to molecules such as antibodies, peptides,and small molecules. The preferred target genes/products for use in thisembodiment are the 14815 genes herein identified. In an alternativeembodiment, the invention provides methods for determining the abilityof the test compound to modulate the activity of a 14815 protein throughmodulation of the activity of a downstream effector of a 14815 targetmolecule. For example, the activity of the effector molecule on anappropriate target can be determined, or the binding of the effector toan appropriate target can be determined, as previously described.

[0243] To identify compounds that interfere with the interaction betweenthe target gene product and its cellular or extracellular bindingpartner(s), a reaction mixture containing the target gene product andthe binding partner is prepared, under conditions and for a timesufficient, to allow the two products to form complex. In order to testan inhibitory agent, the reaction mixture is provided in the presenceand absence of the test compound. The test compound can be initiallyincluded in the reaction mixture, or can be added at a time subsequentto the addition of the target gene and its cellular or extracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe target gene product and the cellular or extracellular bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of thetarget gene product and the interactive binding partner. Additionally,complex formation within reaction mixtures containing the test compoundand normal target gene product can also be compared to complex formationwithin reaction mixtures containing the test compound and mutant targetgene product. This comparison can be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal target gene products.

[0244] These assays can be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring either the target geneproduct or the binding partner onto a solid phase, and detectingcomplexes anchored on the solid phase at the end of the reaction. Inhomogeneous assays, the entire reaction is carried out in a liquidphase. In either approach, the order of addition of reactants can bevaried to obtain different information about the compounds being tested.For example, test compounds that interfere with the interaction betweenthe target gene products and the binding partners, e.g., by competition,can be identified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

[0245] In a heterogeneous assay system, either the target gene productor the interactive cellular or extracellular binding partner, isanchored onto a solid surface (e.g., a microtiter plate), while thenon-anchored species is labeled, either directly or indirectly. Theanchored species can be immobilized by non-covalent or covalentattachments. Alternatively, an immobilized antibody specific orselective for the species to be anchored can be used to anchor thespecies to the solid surface.

[0246] In order to conduct the assay, the partner of the immobilizedspecies is exposed to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific or selective forthe initially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with, e.g., a labeled anti-Igantibody). Depending upon the order of addition of reaction components,test compounds that inhibit complex formation or that disrupt preformedcomplexes can be detected.

[0247] Alternatively, the reaction can be conducted in a liquid phase inthe presence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific or selective for one of the bindingcomponents to anchor any complexes formed in solution, and a labeledantibody specific or selective for the other partner to detect anchoredcomplexes. Again, depending upon the order of addition of reactants tothe liquid phase, test compounds that inhibit complex or that disruptpreformed complexes can be identified.

[0248] In an alternate embodiment of the invention, a homogeneous assaycan be used. For example, a preformed complex of the target gene productand the interactive cellular or extracellular binding partner product isprepared in that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

[0249] In yet another aspect, the 14815 proteins can be used as “baitproteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S.Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with 14815 (“14815-binding proteins” or “14815-bp”) and areinvolved in 14815 activity. Such 14815-bps can be activators orinhibitors of signals by the 14815 proteins or 14815 targets as, forexample, downstream elements of a 14815-mediated signaling pathway.

[0250] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a 14815 protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. (Alternatively the: 14815 protein can bethe fused to the activator domain.) If the “bait” and the “prey”proteins are able to interact, in vivo, forming a 14815-dependentcomplex, the DNA-binding and activation domains of the transcriptionfactor are brought into close proximity. This proximity allowstranscription of a reporter gene (e.g., lacZ) which is operably linkedto a transcriptional regulatory site responsive to the transcriptionfactor. Expression of the reporter gene can be detected and cellcolonies containing the functional transcription factor can be isolatedand used to obtain the cloned gene which encodes the protein whichinteracts with the 14815 protein.

[0251] In another embodiment, modulators of 14815 expression areidentified. For example, a cell or cell free mixture is contacted with acandidate compound and the expression of 14815 mRNA or protein evaluatedrelative to the level of expression of 14815 mRNA or protein in theabsence of the candidate compound. When expression of 14815 mRNA orprotein is greater in the presence of the candidate compound than in itsabsence, the candidate compound is identified as a stimulator of 14815mRNA or protein expression. Alternatively, when expression of 14815 mRNAor protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of 14815 mRNA or protein expression. Thelevel of 14815 mRNA or protein expression can be determined by methodsdescribed herein for detecting 14815 mRNA or protein.

[0252] In another aspect, the invention pertains to a combination of twoor more of the assays described herein. For example, a modulating agentcan be identified using a cell-based or a cell free assay, and theability of the agent to modulate the activity of a 14815 protein can beconfirmed in vivo, e.g., in an animal such as an animal model foraberrant or deficient kinase function or expression.

[0253] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a 14815 modulating agent, an antisense 14815 nucleic acidmolecule, a 14815-specific antibody, or a 14815-binding partner) in anappropriate animal model to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be used for treatments as described herein.

[0254] Detection Assays

[0255] Portions or fragments of the nucleic acid sequences identifiedherein can be used as polynucleotide reagents. For example, thesesequences can be used to: (i) map their respective genes on a chromosomee.g., to locate gene regions associated with genetic disease or toassociate 14815 with a disease; (ii) identify an individual from aminute biological sample (tissue typing); and (iii) aid in forensicidentification of a biological sample. These applications are describedin the subsections below.

[0256] Chromosome Mapping

[0257] The 14815 nucleotide sequences or portions thereof can be used tomap the location of the 14815 genes on a chromosome. This process iscalled chromosome mapping. Chromosome mapping is useful in correlatingthe 14815 sequences with genes associated with disease.

[0258] Briefly, 14815 genes can be mapped to chromosomes by preparingPCR primers (preferably 15-25 bp in length) from the 14815 nucleotidesequences. These primers can then be used for PCR screening of somaticcell hybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the 14815 sequences willyield an amplified fragment.

[0259] A panel of somatic cell hybrids in which each cell line containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, can allow easy mapping ofindividual genes to specific human chromosomes. (D'Eustachio et al.(1983) Science 220:919-924).

[0260] Other mapping strategies e.g., in situ hybridization (describedin Fan et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries can be used to map14815 to a chromosomal location.

[0261] Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results at a reasonable amount of time. For a review of thistechnique, see Verma et al. (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York).

[0262] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0263] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inMcKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland et al. (1987)Nature, 325:783-787.

[0264] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with the 14815 gene,can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

[0265] Tissue Typing

[0266] 14815 sequences can be used to identify individuals frombiological samples using, e.g., restriction fragment length polymorphism(RFLP). In this technique, an individual's genomic DNA is digested withone or more restriction enzymes, the fragments separated, e.g., in aSouthern blot, and probed to yield bands for identification. Thesequences of the present invention are useful as additional DNA markersfor RFLP (described in U.S. Pat. No. 5,272,057).

[0267] Furthermore, the sequences of the present invention can also beused to determine the actual base-by-base DNA sequence of selectedportions of an individual's genome. Thus, the 14815 nucleotide sequencesdescribed herein can be used to prepare two PCR primers from the 5′ and3′ ends of the sequences. These primers can then be used to amplify anindividual's DNA and subsequently sequence it. Panels of correspondingDNA sequences from individuals, prepared in this manner, can provideunique individual identifications, as each individual will have a uniqueset of such DNA sequences due to allelic differences.

[0268] Allelic variation occurs to some degree in the coding regions ofthese sequences, and to a greater degree in the noncoding regions. Eachof the sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1 can provide positiveindividual identification with a panel of perhaps 10 to 1,000 primerswhich each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NO:3 are used, amore appropriate number of primers for positive individualidentification would be 500-2,000.

[0269] If a panel of reagents from 14815 nucleotide sequences describedherein is used to generate a unique identification database for anindividual, those same reagents can later be used to identify tissuefrom that individual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

[0270] Use of Partial 14815 Sequences in Forensic Biology

[0271] DNA-based identification techniques can also be used in forensicbiology. To make such an identification, PCR technology can be used toamplify DNA sequences taken from very small biological samples such astissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, orsemen found at a crime scene. The amplified sequence can then becompared to a standard, thereby allowing identification of the origin ofthe biological sample.

[0272] The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 (e.g., fragments derivedfrom the noncoding regions of SEQ ID NO:1 having a length of at least 20bases, preferably at least 30 bases) are particularly appropriate forthis use.

[0273] The 14815 nucleotide sequences described herein can further beused to provide polynucleotide reagents, e.g., labeled or labelableprobes which can be used in, for example, an in situ hybridizationtechnique, to identify a specific tissue. This can be very useful incases where a forensic pathologist is presented with a tissue of unknownorigin. Panels of such 14815 probes can be used to identify tissue byspecies and/or by organ type.

[0274] In a similar fashion, these reagents, e.g., 14815 primers orprobes can be used to screen tissue culture for contamination (i.e.screen for the presence of a mixture of different types of cells in aculture).

[0275] Predictive Medicine

[0276] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays, and monitoringclinical trials are used for prognostic (predictive) purposes to therebytreat an individual.

[0277] Generally, the invention provides, a method of determining if asubject is at risk for a disorder related to a lesion in or themisexpression of a gene which encodes 14815.

[0278] Such disorders include, e.g., a disorder associated with themisexpression of 14815 gene; a disorder of the cellular proliferativeand/or differentiative system, cell signaling system or nervous system.

[0279] The method includes one or more of the following:

[0280] detecting, in a tissue of the subject, the presence or absence ofa mutation which affects the expression of the 14815 gene, or detectingthe presence or absence of a mutation in a region which controls theexpression of the gene, e.g., a mutation in the 5′ control region;

[0281] detecting, in a tissue of the subject, the presence or absence ofa mutation which alters the structure of the 14815 gene;

[0282] detecting, in a tissue of the subject, the misexpression of the14815 gene, at the mRNA level, e.g., detecting a non-wild type level ofan mRNA;

[0283] detecting, in a tissue of the subject, the misexpression of thegene, at the protein level, e.g., detecting a non-wild type level of a14815 polypeptide.

[0284] In preferred embodiments the method includes: ascertaining theexistence of at least one of: a deletion of one or more nucleotides fromthe 14815 gene; an insertion of one or more nucleotides into the gene, apoint mutation, e.g., a substitution of one or more nucleotides of thegene, a gross chromosomal rearrangement of the gene, e.g., atranslocation, inversion, or deletion.

[0285] For example, detecting the genetic lesion can include: (i)providing a probe/primer including an oligonucleotide containing aregion of nucleotide sequence which hybridizes to a sense or antisensesequence from SEQ ID NO:1, or naturally occurring mutants thereof or 5′or 3′ flanking sequences naturally associated with the 14815 gene; (ii)exposing the probe/primer to nucleic acid of the tissue; and detecting,by hybridization, e.g., in situ hybridization, of the probe/primer tothe nucleic acid, the presence or absence of the genetic lesion.

[0286] In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of the 14815 gene; the presence of anon-wild type splicing pattern of a messenger RNA transcript of thegene; or a non-wild type level of 14815.

[0287] Methods of the invention can be used prenatally or to determineif a subject's offspring will be at risk for a disorder.

[0288] In preferred embodiments the method includes determining thestructure of a 14815 gene, an abnormal structure being indicative ofrisk for the disorder.

[0289] In preferred embodiments the method includes contacting a samplefrom the subject with an antibody to the 14815 protein or a nucleicacid, which hybridizes specifically with the gene. These and otherembodiments are discussed below.

[0290] Diagnostic and Prognostic Assays

[0291] The presence, level, or absence of 14815 protein or nucleic acidin a biological sample can be evaluated by obtaining a biological samplefrom a test subject and contacting the biological sample with a compoundor an agent capable of detecting 14815 protein or nucleic acid (e.g.,mRNA, genomic DNA) that encodes 14815 protein such that the presence of14815 protein or nucleic acid is detected in the biological sample. Theterm “biological sample” includes tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. A preferred biological sample is serum. The level ofexpression of the 14815 gene can be measured in a number of ways,including, but not limited to: measuring the mRNA encoded by the 14815genes; measuring the amount of protein encoded by the 14815 genes; ormeasuring the activity of the protein encoded by the 14815 genes.

[0292] The level of mRNA corresponding to the 14815 gene in a cell canbe determined both by in situ and by in vitro formats.

[0293] The isolated mRNA can be used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onepreferred diagnostic method for the detection of mRNA levels involvescontacting the isolated mRNA with a nucleic acid molecule (probe) thatcan hybridize to the mRNA encoded by the gene being detected. Thenucleic acid probe can be, for example, a full-length 14815 nucleicacid, such as the nucleic acid of SEQ ID NO:1, or a portion thereof,such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to 14815 mRNA or genomic DNA. Other suitable probesfor use in the diagnostic assays are described herein.

[0294] In one format, mRNA (or cDNA) is immobilized on a surface andcontacted with the probes, for example by running the isolated mRNA onan agarose gel and transferring the mRNA from the gel to a membrane,such as nitrocellulose. In an alternative format, the probes areimmobilized on a surface and the mRNA (or cDNA) is contacted with theprobes, for example, in a two-dimensional gene chip array. A skilledartisan can adapt known mRNA detection methods for use in detecting thelevel of mRNA encoded by the 14815 genes.

[0295] The level of mRNA in a sample that is encoded by one of 14815 canbe evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis(1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991)Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al.,(1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al., (1988) Bio/Technology 6:1197), rolling circlereplication (Lizardi et al., U.S. Pat. No. 5,854,033) or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques known in the art. As used herein,amplification primers are defined as being a pair of nucleic acidmolecules that can anneal to 5′ or 3′ regions of a gene (plus and minusstrands, respectively, or vice-versa) and contain a short region inbetween. In general, amplification primers are from about 10 to 30nucleotides in length and flank a region from about 50 to 200nucleotides in length. Under appropriate conditions and with appropriatereagents, such primers permit the amplification of a nucleic acidmolecule comprising the nucleotide sequence flanked by the primers.

[0296] For in situ methods, a cell or tissue sample can beprepared/processed and immobilized on a support, typically a glassslide, and then contacted with a probe that can hybridize to mRNA thatencodes the 14815 gene being analyzed.

[0297] In another embodiment, the methods further contacting a controlsample with a compound or agent capable of detecting 14815 mRNA, orgenomic DNA, and comparing the presence of 14815 mRNA or genomic DNA inthe control sample with the presence of 14815 mRNA or genomic DNA in thetest sample.

[0298] A variety of methods can be used to determine the level ofprotein encoded by 14815. In general, these methods include contactingan agent that selectively binds to the protein, such as an antibody witha sample, to evaluate the level of protein in the sample. In a preferredembodiment, the antibody bears a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with a detectablesubstance. Examples of detectable substances are provided herein.

[0299] The detection methods can be used to detect 14815 protein in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of 14815 protein include enzyme linked immunosorbent assays(ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay(EIA), radioimmunoassay (RIA), and Western blot analysis. In vivotechniques for detection of 14815 protein include introducing into asubject a labeled anti-14815 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

[0300] In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting 14815protein, and comparing the presence of 14815 protein in the controlsample with the presence of 14815 protein in the test sample.

[0301] The invention also includes kits for detecting the presence of14815 in a biological sample. For example, the kit can include acompound or agent capable of detecting 14815 protein or mRNA in abiological sample; and a standard. The compound or agent can be packagedin a suitable container. The kit can further comprise instructions forusing the kit to detect 14815 protein or nucleic acid.

[0302] For antibody-based kits, the kit can include: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide corresponding to a marker of the invention; and, optionally,(2) a second, different antibody which binds to either the polypeptideor the first antibody and is conjugated to a detectable agent.

[0303] For oligonucleotide-based kits, the kit can include: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also includes a buffering agent, apreservative, or a protein stabilizing agent. The kit can also includescomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

[0304] The diagnostic methods described herein can identify subjectshaving, or at risk of developing, a disease or disorder associated withmisexpressed or aberrant or unwanted 14815 expression or activity. Asused herein, the term “unwanted” includes an unwanted phenomenoninvolved in a biological response such as pain or deregulated cellproliferation.

[0305] In one embodiment, a disease or disorder associated with aberrantor unwanted 14815 expression or activity is identified. A test sample isobtained from a subject and 14815 protein or nucleic acid (e.g., mRNA orgenomic DNA) is evaluated, wherein the level, e.g., the presence orabsence, of 14815 protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted 14815 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest, including a biological fluid (e.g., serum), cell sample, ortissue.

[0306] The prognostic assays described herein can be used to determinewhether a subject can be administered an agent (e.g., an agonist,antagonist, peptidomimetic, protein, peptide, nucleic acid, smallmolecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted 14815 expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a cellular proliferative and/ordifferentiative disorder, neurological disorder, liver disorder,apoptotic disorder, metabolic disorder or hormonal disorder.

[0307] The methods of the invention can also be used to detect geneticalterations in a 14815 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation in14815 protein activity or nucleic acid expression, such as a cellularproliferative and/or differentiative disorder, neurological disorder,liver disorder, apoptotic disorder, metabolic disorder or hormonaldisorder. In preferred embodiments, the methods include detecting, in asample from the subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a 14815-protein, or the mis-expression of the 14815gene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a 14815 gene; 2) an addition of one or morenucleotides to a 14815 gene; 3) a substitution of one or morenucleotides of a 14815 gene, 4) a chromosomal rearrangement of a 14815gene; 5) an alteration in the level of a messenger RNA transcript of a14815 gene, 6) aberrant modification of a 14815 gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a 14815 gene, 8)a non-wild type level of a 14815-protein, 9) allelic loss of a 14815gene, and 10) inappropriate post-translational modification of a14815-protein.

[0308] An alteration can be detected without a probe/primer in apolymerase chain reaction, such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR), the latter of whichcan be particularly useful for detecting point mutations in the14815-gene. This method can include the steps of collecting a sample ofcells from a subject, isolating nucleic acid (e.g., genomic, mRNA orboth) from the sample, contacting the nucleic acid sample with one ormore primers which specifically hybridize to a 14815 gene underconditions such that hybridization and amplification of the 14815 gene(if present) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein. Alternatively, other amplificationmethods described herein or known in the art can be used.

[0309] In another embodiment, mutations in a 14815 gene from a samplecell can be identified by detecting alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined, e.g., by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0310] In other embodiments, genetic mutations in 14815 can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, two dimensional arrays, e.g., chip based arrays. Such arraysinclude a plurality of addresses, each of which is positionallydistinguishable from the other. A different probe is located at eachaddress of the plurality. The arrays can have a high density ofaddresses, e.g., can contain hundreds or thousands of oligonucleotidesprobes (Cronin et al. (1996) Human Mutation 7: 244-255; Kozal et al.(1996) Nature Medicine 2: 753-759). For example, genetic mutations in14815 can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

[0311] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the 14815gene and detect mutations by comparing the sequence of the sample 14815with the corresponding wild-type (control) sequence. Automatedsequencing procedures can be utilized when performing the diagnosticassays (Naeve et al. (1995) Biotechniques 19:448-53), includingsequencing by mass spectrometry.

[0312] Other methods for detecting mutations in the 14815 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[0313] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in 14815 cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662; U.S. Pat. No. 5,459,039).

[0314] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in 14815 genes. For example, singlestrand conformation polymorphism (SSCP) can be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control 14815 nucleic acids will be denatured and allowed torenature. The secondary structure of single-stranded nucleic acidsvaries according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments can be labeled or detected with labeledprobes. The sensitivity of the assay can be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet 7:5).

[0315] In yet another embodiment, the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

[0316] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. NatlAcad. Sci USA 86:6230).

[0317] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification can be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification can carry the mutation of interest in the center of themolecule (so that amplification depends on differential hybridization)(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner (1993) Tibtech11:238). In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It isanticipated that in certain embodiments amplification can also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci USA 88:189-93). In such cases, ligation will occur only ifthere is a perfect match at the 3′ end of the 5′ sequence making itpossible to detect the presence of a known mutation at a specific siteby looking for the presence or absence of amplification.

[0318] The methods described herein can be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which can beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga 14815 gene.

[0319] Use of 14815 Molecules as Surrogate Markers

[0320] The 14815 molecules of the invention are also useful as markersof disorders or disease states, as markers for precursors of diseasestates, as markers for predisposition of disease states, as markers ofdrug activity, or as markers of the pharmacogenomic profile of asubject. Using the methods described herein, the presence, absenceand/or quantity of the 14815 molecules of the invention can be detected,and can be correlated with one or more biological states in vivo. Forexample, the 14815 molecules of the invention can serve as surrogatemarkers for one or more disorders or disease states or for conditionsleading up to disease states. As used herein, a “surrogate marker” is anobjective biochemical marker which correlates with the absence orpresence of a disease or disorder, or with the progression of a diseaseor disorder (e.g., with the presence or absence of a tumor). Thepresence or quantity of such markers is independent of the disease.Therefore, these markers can serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease can be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection can be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[0321] The 14815 molecules of the invention are also useful aspharmacodynamic markers. As used herein, a “pharmacodynamic marker” isan objective biochemical marker which correlates specifically with drugeffects. The presence or quantity of a pharmacodynamic marker is notrelated to the disease state or disorder for which the drug is beingadministered; therefore, the presence or quantity of the marker isindicative of the presence or activity of the drug in a subject. Forexample, a pharmacodynamic marker can be indicative of the concentrationof the drug in a biological tissue, in that the marker is eitherexpressed or transcribed or not expressed or transcribed in that tissuein relationship to the level of the drug. In this fashion, thedistribution or uptake of the drug can be monitored by thepharmacodynamic marker. Similarly, the presence or quantity of thepharmacodynamic marker can be related to the presence or quantity of themetabolic product of a drug, such that the presence or quantity of themarker is indicative of the relative breakdown rate of the drug in vivo.Pharmacodynamic markers are of particular use in increasing thesensitivity of detection of drug effects, particularly when the drug isadministered in low doses. Since even a small amount of a drug can besufficient to activate multiple rounds of marker (e.g., a 14815 marker)transcription or expression, the amplified marker can be in a quantitywhich is more readily detectable than the drug itself. Also, the markercan be more easily detected due to the nature of the marker itself; forexample, using the methods described herein, anti-14815 antibodies canbe employed in an immune-based detection system for a 14815 proteinmarker, or 14815-specific radiolabeled probes can be used to detect a14815 mRNA marker. Furthermore, the use of a pharmacodynamic marker canoffer mechanism-based prediction of risk due to drug treatment beyondthe range of possible direct observations. Examples of the use ofpharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No.6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238;Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andNicolau (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[0322] The 14815 molecules of the invention are also useful aspharmacogenomic markers. As used herein, a “pharmacogenomic marker” isan objective biochemical marker which correlates with a specificclinical drug response or susceptibility in a subject (see, e.g., McLeodet al. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity ofthe pharmacogenomic marker is related to the predicted response of thesubject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of one or morepharmacogenomic markers in a subject, a drug therapy which is mostappropriate for the subject, or which is predicted to have a greaterdegree of success, can be selected. For example, based on the presenceor quantity of RNA, or protein (e.g., 14815 protein or RNA) for specifictumor markers in a subject, a drug or course of treatment can beselected that is optimized for the treatment of the specific tumorlikely to be present in the subject. Similarly, the presence or absenceof a specific sequence mutation in 14815 DNA can correlate with a 14815drug response. The use of pharmacogenomic markers therefore permits theapplication of the most appropriate treatment for each subject withouthaving to administer the therapy.

[0323] Pharmaceutical Compositions

[0324] The nucleic acid and polypeptides, fragments thereof, as well asanti-14815 antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositions. Suchcompositions typically include the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

[0325] A pharmaceutical composition is formulated to be compatible withits intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral, transdermal (e.g. topical), transmucosal (e.g.,inhalation of aerosol or absorption of eye drop), and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0326] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0327] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0328] Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0329] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0330] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0331] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0332] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0333] It is advantageous to formulate oral or parenteral compositionsin dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

[0334] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ ED₅₀. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects can be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0335] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

[0336] As defined herein, a therapeutically effective amount of proteinor polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout I to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody, unconjugated or conjugated asdescribed herein, can include a single treatment or, preferably, caninclude a series of treatments.

[0337] For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

[0338] The present invention encompasses agents which modulateexpression or activity. An agent can, for example, be a small molecule.For example, such small molecules include, but are not limited to,peptides, peptidomimetics (e.g., peptoids), amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (i.e.,. includingheteroorganic and organometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds.

[0339] Exemplary doses include milligram or microgram amounts of thesmall molecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher can, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

[0340] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0341] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0342] Methods of Treatment:

[0343] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with aberrant or unwanted14815 expression or activity. As used herein, the term “treatment” isdefined as the application or administration of a therapeutic agent to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. A therapeutic agent includes, but is notlimited to, small molecules, peptides, antibodies, ribozymes andantisense oligonucleotides.

[0344] With regards to both prophylactic and therapeutic methods oftreatment, such treatments can be specifically tailored or modified,based on knowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”.) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the 14815 molecules ofthe present invention or 14815 modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and not to provide this treatment topatients who will experience toxic drug-related side effects.

[0345] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with an aberrant orunwanted 14815 expression or activity, by administering to the subject a14815 or an agent which modulates 14815 expression or at least one 14815activity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted 14815 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe 14815 aberrance, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type of14815 aberrance, for example, a 14815, 14815 agonist or 14815 antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

[0346] It is possible that some 14815 disorders can be caused, at leastin part, by an abnormal level of gene product, or by the presence of agene product exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of such gene products would bring about theamelioration of disorder symptoms.

[0347] The 14815 molecules can act as novel diagnostic targets andtherapeutic agents for controlling one or more of neurologicaldisorders, hepatic disorders, cellular proliferative and/ordifferentiative disorders, inflammatory disorders, apoptotic disorders,metabolic disorders and hormonal disorders, all of which are describedabove.

[0348] As discussed, successful treatment of 14815 disorders can bebrought about by techniques that serve to inhibit the expression oractivity of target gene products. For example, compounds, e.g., an agentidentified using an assays described above, that proves to exhibitnegative modulatory activity, can be used in accordance with theinvention to prevent and/or ameliorate symptoms of 14815 disorders. Suchmolecules can include, but are not limited to peptides, phosphopeptides,small organic or inorganic molecules, or antibodies (including, forexample, polyclonal, monoclonal, humanized, human, anti-idiotypic,chimeric or single chain antibodies, and Fab, F(ab′)₂ and Fab expressionlibrary fragments, scFV molecules, and epitope-binding fragmentsthereof).

[0349] Further, antisense and ribozyme molecules that inhibit expressionof the target gene can also be used in accordance with the invention toreduce the level of target gene expression, thus effectively reducingthe level of target gene activity. Still further, triple helix moleculescan be utilized in reducing the level of target gene activity.Antisense, ribozyme and triple helix molecules are discussed above.

[0350] It is possible that the use of antisense, ribozyme, and/or triplehelix molecules to reduce or inhibit mutant gene expression can alsoreduce or inhibit the transcription (triple helix) and/or translation(antisense, ribozyme) of mRNA produced by normal target gene alleles,such that the concentration of normal target gene product present can belower than is necessary for a normal phenotype. In such cases, nucleicacid molecules that encode and express target gene polypeptidesexhibiting normal target gene activity can be introduced into cells viagene therapy method. Alternatively, in instances in that the target geneencodes an extracellular protein, it can be preferable to co-administernormal target gene protein into the cell or tissue in order to maintainthe requisite level of cellular or tissue target gene activity.

[0351] Another method by which nucleic acid molecules can be utilized intreating or preventing a disease characterized by 14815 expression isthrough the use of aptamer molecules specific for 14815 protein.Aptamers are nucleic acid molecules having a tertiary structure whichpermits them to specifically or selectively bind to protein ligands(see, e.g., Osborne et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; andPatel (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid moleculescan in many cases be more conveniently introduced into target cells thantherapeutic protein molecules can be, aptamers offer a method by which14815 protein activity can be specifically decreased without theintroduction of drugs or other molecules which can have pluripotenteffects.

[0352] Antibodies can be generated that are both specific for targetgene product and that reduce target gene product activity. Suchantibodies can, therefore, by administered in instances whereby negativemodulatory techniques are appropriate for the treatment of 14815disorders. For a description of antibodies, see the Antibody sectionabove.

[0353] In circumstances wherein injection of an animal or a humansubject with a 14815 protein or epitope for stimulating antibodyproduction is harmful to the subject, it is possible to generate animmune response against 14815 through the use of anti-idiotypicantibodies (see, for example, Herlyn (1999) Ann Med 31:66-78; andBhattacharya-Chatterjee and Foon (1998) Cancer Treat Res. 94:51-68). Ifan anti-idiotypic antibody is introduced into a mammal or human subject,it should stimulate the production of anti-anti-idiotypic antibodies,which should be specific to the 14815 protein. Vaccines directed to adisease characterized by 14815 expression can also be generated in thisfashion.

[0354] In instances where the target antigen is intracellular and wholeantibodies are used, internalizing antibodies can be preferred.Lipofectin or liposomes can be used to deliver the antibody or afragment of the Fab region that binds to the target antigen into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is preferred. For example,peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizingantibodies that bind to intracellular target antigens can also beadministered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (see e.g., Marasco et al.(1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[0355] The identified compounds that inhibit target gene expression,synthesis and/or activity can be administered to a patient attherapeutically effective doses to prevent, treat or ameliorate 14815disorders. A therapeutically effective dose refers to that amount of thecompound sufficient to result in amelioration of symptoms of thedisorders. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures as described above.

[0356] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies. preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

[0357] Another example of determination of effective dose for anindividual is the ability to directly assay levels of “free” and “bound”compound in the serum of the test subject. Such assays can utilizeantibody mimics and/or “biosensors” that have been created throughmolecular imprinting techniques. The compound which is able to modulate14815 activity is used as a template, or “imprinting molecule”, tospatially organize polymerizable monomers prior to their polymerizationwith catalytic reagents. The subsequent removal of the imprintedmolecule leaves a polymer matrix which contains a repeated “negativeimage” of the compound and is able to selectively rebind the moleculeunder biological assay conditions. A detailed review of this techniquecan be seen in Ansell et al (1996) Current Opinion in Biotechnology7:89-94 and in Shea (1994) Trends in Polymer Science 2:166-173. Such“imprinted” affinity matrixes are amenable to ligand-binding assays,whereby the immobilized monoclonal antibody component is replaced by anappropriately imprinted matrix. An example of the use of such matrixesin this way can be seen in Vlatakis et al (1993) Nature 361:645-647.Through the use of isotope-labeling, the “free” concentration ofcompound which modulates the expression or activity of 14815 can bereadily monitored and used in calculations of IC₅₀.

[0358] Such “imprinted” affinity matrixes can also be designed toinclude fluorescent groups whose photon-emitting properties measurablychange upon local and selective binding of target compound. Thesechanges can be readily assayed in real time using appropriate fiberopticdevices, in turn allowing the dose in a test subject to be quicklyoptimized based on its individual IC₅₀. An rudimentary example of such a“biosensor” is discussed in Kriz et al (1995) Analytical Chemistry67:2142-2144.

[0359] Another aspect of the invention pertains to methods of modulating14815 expression or activity for therapeutic purposes. Accordingly, inan exemplary embodiment, the modulatory method of the invention involvescontacting a cell with a 14815 or agent that modulates one or more ofthe activities of 14815 protein activity associated with the cell. Anagent that modulates 14815 protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a 14815 protein (e.g., a 14815 substrate orreceptor), a 14815 antibody, a 14815 agonist or antagonist, apeptidomimetic of a 14815 agonist or antagonist, or other smallmolecule.

[0360] In one embodiment, the agent stimulates one or 14815 activities.Examples of such stimulatory agents include active 14815 protein and anucleic acid molecule encoding 14815. In another embodiment, the agentinhibits one or more 14815 activities. Examples of such inhibitoryagents include antisense 14815 nucleic acid molecules, anti-14815antibodies, and 14815 inhibitors. These modulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a 14815 protein ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g., upregulates or down regulates) 14815 expression or activity. In anotherembodiment, the method involves administering a 14815 protein or nucleicacid molecule as therapy to compensate for reduced, aberrant, orunwanted 14815 expression or activity.

[0361] Stimulation of 14815 activity is desirable in situations in which14815 is abnormally downregulated and/or in which increased 14815activity is likely to have a beneficial effect. For example, stimulationof 14815 activity is desirable in situations in which a 14815 isdownregulated and/or in which increased 14815 activity is likely to havea beneficial effect. Likewise, inhibition of 14815 activity is desirablein situations in which 14815 is abnormally upregulated and/or in whichdecreased 14815 activity is likely to have a beneficial effect.

[0362] Pharmacogenomics

[0363] The 14815 molecules of the present invention, as well as agents,or modulators which have a stimulatory or inhibitory effect on 14815activity (e.g., 14815 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) 14815-associated disorders (e.g.,aberrant or deficient kinase function or expression) associated withaberrant or unwanted 14815 activity. In conjunction with such treatment,pharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) can be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician canconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a 14815 molecule or 14815modulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a 14815 molecule or 14815 modulator.

[0364] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, for example, Eichelbaum et al.(1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder et al.(1997) Clin. Chem. 43:254-266. In general, two types of pharmacogeneticconditions can be differentiated. Genetic conditions transmitted as asingle factor altering the way drugs act on the body (altered drugaction) or genetic conditions transmitted as single factors altering theway the body acts on drugs (altered drug metabolism). Thesepharmacogenetic conditions can occur either as rare genetic defects oras naturally-occurring polymorphisms. For example, glucose-6-phosphatedehydrogenase deficiency (G6PD) is a common inherited enzymopathy inwhich the main clinical complication is haemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

[0365] One pharmacogenomics approach to identifying genes that predictdrug response, known as “a genome-wide association”, relies primarily ona high-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP can occur once per every 1000 bases of DNA. ASNP can be involved in a disease process, however, the vast majority cannot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that can becommon among such genetically similar individuals.

[0366] Alternatively, a method termed the “candidate gene approach”, canbe utilized to identify genes that predict drug response. According tothis method, if a gene that encodes a drug's target is known (e.g., a14815 protein of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

[0367] Alternatively, a method termed the “gene expression profiling”,can be utilized to identify genes that predict drug response. Forexample, the gene expression of an animal dosed with a drug (e.g., a14815 molecule or 14815 modulator of the present invention) can give anindication whether gene pathways related to toxicity have been turnedon.

[0368] Information generated from more than one of the abovepharmacogenomics approaches can be used to determine appropriate dosageand treatment regimens for prophylactic or therapeutic treatment of anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with a14815 molecule or 14815 modulator, such as a modulator identified by oneof the exemplary screening assays described herein.

[0369] The present invention further provides methods for identifyingnew agents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the 14815 genes of the present invention, wherein theseproducts can be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by the 14815genes of the present invention can be used as a basis for identifyingagents for overcoming agent resistance. By blocking the activity of oneor more of the resistance proteins, target cells, e.g., human cells,will become sensitive to treatment with an agent to which the unmodifiedtarget cells were resistant.

[0370] Monitoring the influence of agents (e.g., drugs) on theexpression or activity of a 14815 protein can be applied in clinicaltrials. For example, the effectiveness of an agent determined by ascreening assay as described herein to increase 14815 gene expression,protein levels, or upregulate 14815 activity, can be monitored inclinical trials of subjects exhibiting decreased 14815 gene expression,protein levels, or downregulated 14815 activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decrease14815 gene expression, protein levels, or downregulate 14815 activity,can be monitored in clinical trials of subjects exhibiting increased14815 gene expression, protein levels, or upregulated 14815 activity. Insuch clinical trials, the expression or activity of a 14815 gene, andpreferably, other genes that have been implicated in, for example, akinase-associated or another 14815-associated disorder can be used as a“read out” or markers of the phenotype of a particular cell.

[0371] Other Embodiments

[0372] In another aspect, the invention features a method of analyzing aplurality of capture probes. The method is useful, e.g., to analyze geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence, wherein the capture probes are from acell or subject which expresses 14815 or from a cell or subject in whicha 14815 mediated response has been elicited; contacting the array with a14815 nucleic acid (preferably purified), a 14815 polypeptide(preferably purified), or an anti-14815 antibody, and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization with a capture probe at an address of the plurality,is detected, e.g., by a signal generated from a label attached to the14815 nucleic acid, polypeptide, or antibody.

[0373] The capture probes can be a set of nucleic acids from a selectedsample, e.g., a sample of nucleic acids derived from a control ornon-stimulated tissue or cell.

[0374] The method can include contacting the 14815 nucleic acid,polypeptide, or antibody with a first array having a plurality ofcapture probes and a second array having a different plurality ofcapture probes. The results of each hybridization can be compared, e.g.,to analyze differences in expression between a first and second sample.The first plurality of capture probes can be from a control sample,e.g., a wild type, normal, or non-diseased, non-stimulated, sample,e.g., a biological fluid, tissue, or cell sample. The second pluralityof capture probes can be from an experimental sample, e.g., a mutanttype, at risk, disease-state or disorder-state, or stimulated, sample,e.g., a biological fluid, tissue, or cell sample.

[0375] The plurality of capture probes can be a plurality of nucleicacid probes each of which specifically hybridizes, with an allele of14815. Such methods can be used to diagnose a subject, e.g., to evaluaterisk for a disease or disorder, to evaluate suitability of a selectedtreatment for a subject, to evaluate whether a subject has a disease ordisorder.

[0376] The method can be used to detect SNPs, as described above.

[0377] In another aspect, the invention features, a method of analyzing14815, e.g., analyzing structure, function, or relatedness to othernucleic acid or amino acid sequences. The method includes: providing a14815 nucleic acid or amino acid sequence; comparing the 14815 sequencewith one or more preferably a plurality of sequences from a collectionof sequences, e.g., a nucleic acid or protein sequence database; tothereby analyze 14815.

[0378] The method can include evaluating the sequence identity between a14815 sequence and a database sequence. The method can be performed byaccessing the database at a second site, e.g., over the internet.Preferred databases include GenBank™ and SwissProt.

[0379] In another aspect, the invention features, a set ofoligonucleotides, useful, e.g., for identifying SNP's, or identifyingspecific alleles of 14815. The set includes a plurality ofoligonucleotides, each of which has a different nucleotide at aninterrogation position, e.g., an SNP or the site of a mutation. In apreferred embodiment, the oligonucleotides of the plurality identical insequence with one another (except for differences in length). Theoligonucleotides can be provided with differential labels, such that anoligonucleotide which hybridizes to one allele provides a signal that isdistinguishable from an oligonucleotides which hybridizes to a secondallele.

[0380] The sequences of 14815 molecules are provided in a variety ofmediums to facilitate use thereof. A sequence can be provided as amanufacture, other than an isolated nucleic acid or amino acid molecule,which contains a 14815 molecule. Such a manufacture can provide anucleotide or amino acid sequence, e.g., an open reading frame, in aform which allows examination of the manufacture using means notdirectly applicable to examining the nucleotide or amino acid sequences,or a subset thereof, as they exist in nature or in purified form.

[0381] A 14815 nucleotide or amino acid sequence can be recorded oncomputer readable media. As used herein, “computer readable media”refers to any medium that can be read and accessed directly by acomputer. Such media include, but are not limited to: magnetic storagemedia, such as floppy discs, hard disc storage medium, and magnetictape; optical storage media such as compact disc and CD-ROM; electricalstorage media such as RAM, ROM, EPROM, EEPROM, and the like; and generalhard disks and hybrids of these categories such as magnetic/opticalstorage media. The medium is adapted or configured for having thereon14815 sequence information of the present invention.

[0382] As used herein, the term “electronic apparatus” is intended toinclude any suitable computing or processing apparatus of other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as personal digital assistants(PDAs), cellular phones, pagers, and the like; and local and distributedprocessing systems.

[0383] As used herein, “recorded” refers to a process for storing orencoding information on the electronic apparatus readable medium. Thoseskilled in the art can readily adopt any of the presently known methodsfor recording information on known media to generate manufacturescomprising the 14815 sequence information.

[0384] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereona 14815 nucleotide or amino acid sequence of the present invention. Thechoice of the data storage structure will generally be based on themeans chosen to access the stored information. In addition, a variety ofdata processor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order toobtain computer readable medium having recorded thereon the nucleotidesequence information of the present invention.

[0385] By providing the 14815 nucleotide or amino acid sequences of theinvention in computer readable form, the skilled artisan can routinelyaccess the sequence information for a variety of purposes. For example,one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. A search is used to identify fragments or regions ofthe sequences of the invention which match a particular target sequenceor target motif.

[0386] The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas a kinase-associated or another 14815-associated disease or disorderor a pre-disposition to a kinase-associated or another 14815-associateddisease or disorder, wherein the method comprises the steps ofdetermining 14815 sequence information associated with the subject andbased on the 14815 sequence information, determining whether the subjecthas a kinase-associated or another 14815-associated disease or disorderand/or recommending a particular treatment for the disease, disorder, orpre-disease condition.

[0387] The present invention further provides in an electronic systemand/or in a network, a method for determining whether a subject has akinase-associated or another 14815-associated disease or disorder or apre-disposition to a disease associated with 14815, wherein the methodcomprises the steps of determining 14815 sequence information associatedwith the subject, and based on the 14815 sequence information,determining whether the subject has a kinase-associated or another14815-associated disease or disorder or a pre-disposition to akinase-associated or another 14815-associated disease or disorder,and/or recommending a particular treatment for the disease, disorder, orpre-disease condition. The method may further comprise the step ofreceiving phenotypic information associated with the subject and/oracquiring from a network phenotypic information associated with thesubject.

[0388] The present invention also provides in a network, a method fordetermining whether a subject has a kinase-associated or another14815-associated disease or disorder or a pre-disposition to akinase-associated or another 14815-associated disease or disorder, saidmethod comprising the steps of receiving 14815 sequence information fromthe subject and/or information related thereto, receiving phenotypicinformation associated with the subject, acquiring information from thenetwork corresponding to 14815 and/or corresponding to akinase-associated or another 14815-associated disease or disorder, andbased on one or more of the phenotypic information, the 14815information (e.g., sequence information and/or information relatedthereto), and the acquired information, determining whether the subjecthas a kinase-associated or another 14815-associated disease or disorderor a pre-disposition to a kinase-associated or another 14815-associateddisease or disorder. The method may further comprise the step ofrecommending a particular treatment for the disease, disorder, orpre-disease condition.

[0389] The present invention also provides a business method fordetermining whether a subject has a kinase-associated or another14815-associated disease or disorder or a pre-disposition to akinase-associated or another 14815-associated disease or disorder, saidmethod comprising the steps of receiving information related to 14815(e.g., sequence information and/or information related thereto),receiving phenotypic information associated with the subject, acquiringinformation from the network related to 14815 and/or related to akinase-associated or another 14815-associated disease or disorder, andbased on one or more of the phenotypic information, the 14815information, and the acquired information, determining whether thesubject has a kinase-associated or another 14815-associated disease ordisorder or a pre-disposition to a kinase-associated or another14815-associated disease or disorder. The method may further comprisethe step of recommending a particular treatment for the disease,disorder, or pre-disease condition.

[0390] The invention also includes an array comprising a 14815 sequenceof the present invention. The array can be used to assay expression ofone or more genes in the array. In one embodiment, the array can be usedto assay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 7600 genes can besimultaneously assayed for expression, one of which can be 14815. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

[0391] In addition to such qualitative information, the invention allowsthe quantitation of gene expression. Thus, not only tissue specificity,but also the level of expression of a battery of genes in the tissue ifascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionin that tissue. Thus, one tissue can be perturbed and the effect on geneexpression in a second tissue can be determined. In this context, theeffect of one cell type on another cell type in response to a biologicalstimulus can be determined. In this context, the effect of one cell typeon another cell type in response to a biological stimulus can bedetermined. Such a determination is useful, for example, to know theeffect of cell-cell interaction at the level of gene expression. If anagent is administered therapeutically to treat one cell type but has anundesirable effect on another cell type, the invention provides an assayto determine the molecular basis of the undesirable effect and thusprovides the opportunity to co-administer a counteracting agent orotherwise treat the undesired effect. Similarly, even within a singlecell type, undesirable biological effects can be determined at themolecular level. Thus, the effects of an agent on expression of otherthan the target gene can be ascertained and counteracted.

[0392] In another embodiment, the array can be used to monitor the timecourse of expression of one or more genes in the array. This can occurin various biological contexts, as disclosed herein, for exampledevelopment of a kinase-associated or another 14815-associated diseaseor disorder, progression of kinase-associated or another14815-associated disease or disorder, and processes, such a cellulartransformation associated with the kinase-associated or another14815-associated disease or disorder.

[0393] The array is also useful for ascertaining the effect of theexpression of a gene on the expression of other genes in the same cellor in different cells (e.g., acertaining the effect of 14815 expressionon the expression of other genes). This provides, for example, for aselection of alternate molecular targets for therapeutic intervention ifthe ultimate or downstream target cannot be regulated.

[0394] The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including 14815) that could serve asa molecular target for diagnosis or therapeutic intervention.

[0395] As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. Typical sequence lengths of a targetsequence are from about 10 to 100 amino acids or from about 30 to 300nucleotide residues. However, it is well recognized that commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

[0396] Computer software is publicly available which allows a skilledartisan to access sequence information provided in a computer readablemedium for analysis and comparison to other sequences. A variety ofknown algorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware include, but are not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBI).

[0397] Thus, the invention features a method of making a computerreadable record of a sequence of a 14815 sequence which includesrecording the sequence on a computer readable matrix. In a preferredembodiment the record includes one or more of the following:identification of an ORF; identification of a domain, region, or site;identification of the start of transcription; identification of thetranscription terminator; the full length amino acid sequence of theprotein, or a mature form thereof; the 5′ end of the translated region.

[0398] In another aspect, the invention features a method of analyzing asequence. The method includes: providing a 14815 sequence, or record, incomputer readable form; comparing a second sequence to the 14815sequence; thereby analyzing a sequence. Comparison can include comparingto sequences for sequence identity or determining if one sequence isincluded within the other, e.g., determining if the 14815 sequenceincludes a sequence being compared. In a preferred embodiment the 14815or second sequence is stored on a first computer, e.g., at a first siteand the comparison is performed, read, or recorded on a second computer,e.g., at a second site. E.g., the 14815 or second sequence can be storedin a public or proprietary database in one computer, and the results ofthe comparison performed, read, or recorded on a second computer. In apreferred embodiment the record includes one or more of the following:identification of an ORF; identification of a domain, region, or site;identification of the start of transcription; identification of thetranscription terminator; the full length amino acid sequence of theprotein, or a mature form thereof; the 5′ end of the translated region.

[0399] This invention is further illustrated by the followingexemplification, which should not be construed as limiting.

EXEMPLIFICATION

[0400] Gene Expression Analysis

[0401] Total RNA was prepared from various human tissues by a singlestep extraction method using RNA STAT-60 according to the manufacturer'sinstructions (TelTest, Inc). Each RNA preparation was treated with DNaseI (Ambion) at 37° C. for 1 hour. DNAse I treatment was determined to becomplete if the sample required at least 38 PCR amplification cycles toreach a threshold level of fluorescence using β-2 microglobulin as aninternal amplicon reference. The integrity of the RNA samples followingDNase I treatment was confirmed by agarose gel electrophoresis andethidium bromide staining. After phenol extraction cDNA was preparedfrom the sample using the SUPERSCRIPT™ Choice System following themanufacturer's instructions (GibcoBRL). A negative control of RNAwithout reverse transcriptase was mock reverse transcribed for each RNAsample.

[0402] Human 14815 expression was measured by TaqMan® quantitative PCR(Perkin Elmer Applied Biosystems) in cDNA prepared from a variety ofnormal and diseased (e.g., cancerous) human tissues or cell lines.

[0403] Probes were designed by PrimerExpress software (PE Biosystems)based on the sequence of the human 14815 gene. Each human 14815 geneprobe was labeled using FAM (6-carboxyfluorescein), and theβ2-microglobulin reference probe was labeled with a differentfluorescent dye, VIC. The differential labeling of the target gene andinternal reference gene thus enabled measurement in same well. Forwardand reverse primers and the probes for both β2-microglobulin and targetgene were added to the TaqMan® Universal PCR Master Mix (PE AppliedBiosystems). Although the final concentration of primer and probe couldvary, each was internally consistent within a given experiment. Atypical experiment contained 200 nM of forward and reverse primers plus100 nM probe for β-2 microglobulin and 600 nM forward and reverseprimers plus 200 nM probe for the target gene. TaqMan matrix experimentswere carried out on an ABI PRISM 7700 Sequence Detection System (PEApplied Biosystems). The sequences of the reagents used to identify14815 expression included a forward primer, AAGACTGGGCTGAGTTTGTGAAG, SEQID NO:10; a reverse primer, CCAAGGGATTGCCTACAAACA, SEQ ID NO:11, and aprobe, AACTGCCATGCCTCGAAGACCTGG, SEQ ID NO:12. The thermal cyclerconditions were as follows: hold for 2 min at 50° C. and 10 min at 95°C., followed by two-step PCR for 40 cycles of 95° C. for 15 sec followedby 60° C. for 1 min.

[0404] The following method was used to quantitatively calculate human14815 gene expression in the various tissues relative to β-2microglobulin expression in the same tissue. The threshold cycle (Ct)value is defined as the cycle at which a statistically significantincrease in fluorescence is detected. A lower Ct value is indicative ofa higher mRNA concentration. The Ct value of the human 14815 gene isnormalized by subtracting the Ct value of the β-2 microglobulin gene toobtain a _(Δ)Ct value using the following formula:_(Δ)Ct=Ct_(human 59914 and 59921)−Ct _(β-2) microglobulin Expression isthen calibrated against a cDNA sample showing a comparatively low levelof expression of the human 14815 gene. The _(Δ)Ct value for thecalibrator sample is then subtracted from _(Δ)Ct for each tissue sampleaccording to the following formula:_(ΔΔ)Ct=_(Δ)Ct-_(sample)−_(Δ)Ct-_(calibrator). Relative expression isthen calculated using the arithmetic formula given by 2^(−ΔΔCt).Expression of the target human 14815 gene in each of the tissues testedis then graphically represented as discussed in more detail below.

[0405] The results indicate significant 14815 expression in normalbrain, normal liver and in breast tissue and primary breast tumors, butmuch less in metastases from the primary tumor.

[0406] Expression analysis surveys have found 14815 expression in immunesystem cells and tissues and have studied changes in 14815 expression incells and tissues typically involved in inflammatory disorders. Theresults indicate that certain resting lymphocytes have higher 14815expression than receptor-stimulated lymphocytes. For example, 14815expression in resting CD8+ lymphocytes is eight-fold higher than in CD8+lymphocytes stimulated with anti-CD3 antibodies. In another example,14815 expression in resting normal human lung fibroblasts is three- tofive-fold higher than in normal human lung fibroblasts stimulated withtransforming growth factor beta or tumor necrosis factor alpha. Inanother example, 14815 expression in resting normal human dermalfibroblasts is two- to three-fold higher than in normal human dermalfibroblasts stimulated with transforming growth factor beta orinterleukin-1b. In another example, 14815 expression in restingfibroblast-like synoviocytes is two-fold higher than in fibroblast-likesynoviocytes stimulated with tumor necrosis factor alpha orinterleukin-1. In another example, 14815 expression is two-fold higherin resting normal human bronchial epithelial cells compared withinterleukin-4- or interleukin-13-stimulated normal human bronchialepithelial cells. In a further example, 14815 expression in restingbronchial smooth muscle cells is higher than in bronchial smooth musclecells stimulated with tumor necrosis factor alpha or interferon gamma.

[0407] The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference.

[0408] Equivalents

[0409] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein.

1 12 1 3919 DNA Homo sapiens CDS (183)...(3530) misc_feature 3899 n=a,t, c, or g 1 gacgggctga gactggacgg gacccccggt ctgcagcagc aggtgacagcagcagggaca 60 atgataagga gattggcctg aaggagggac cgtccctccc gcgcgaaaagtcagaaatgg 120 ccaatgaagc ttttgcttat aaaaggaatg cgatgttaat tctggggcattgatgtttta 180 ca atg cct gat caa gat aaa aag gtg aag acc aca gaa aaatca act 227 Met Pro Asp Gln Asp Lys Lys Val Lys Thr Thr Glu Lys Ser Thr1 5 10 15 gat aaa cag caa gaa atc acc atc agg gac tat tca gat ctt aaaaga 275 Asp Lys Gln Gln Glu Ile Thr Ile Arg Asp Tyr Ser Asp Leu Lys Arg20 25 30 ctt cgg tgc ctt ttg aac gtc caa tca agc aaa caa cag ctt cca gcc323 Leu Arg Cys Leu Leu Asn Val Gln Ser Ser Lys Gln Gln Leu Pro Ala 3540 45 att aac ttc gat agt gcc caa aat agc atg acg aag tct gag ccc gcc371 Ile Asn Phe Asp Ser Ala Gln Asn Ser Met Thr Lys Ser Glu Pro Ala 5055 60 atc agg gcg ggt gga cac aga gct cgg ggt cag tgg cat gaa tcc aca419 Ile Arg Ala Gly Gly His Arg Ala Arg Gly Gln Trp His Glu Ser Thr 6570 75 gaa gct gtt gaa ctt gaa aat ttt agt ata aac tac aag aat gag aga467 Glu Ala Val Glu Leu Glu Asn Phe Ser Ile Asn Tyr Lys Asn Glu Arg 8085 90 95 aat ttc agc aaa cat cct cag cgt aaa cta ttt cag gag atc ttt acc515 Asn Phe Ser Lys His Pro Gln Arg Lys Leu Phe Gln Glu Ile Phe Thr 100105 110 gcc ttg gtg aaa aat aga ctc ata agc aga gag tgg gtt aat cga gcc563 Ala Leu Val Lys Asn Arg Leu Ile Ser Arg Glu Trp Val Asn Arg Ala 115120 125 cca tct att cat ttt ctg aga gtg tta atc tgt ctg agg cta cta atg611 Pro Ser Ile His Phe Leu Arg Val Leu Ile Cys Leu Arg Leu Leu Met 130135 140 agg gat cca tgt tat cag gaa ata ctc cat agc ttg ggt ggg att gaa659 Arg Asp Pro Cys Tyr Gln Glu Ile Leu His Ser Leu Gly Gly Ile Glu 145150 155 aac cta gct cag tat atg gag att gta gcc aat gag tac ctc ggc tat707 Asn Leu Ala Gln Tyr Met Glu Ile Val Ala Asn Glu Tyr Leu Gly Tyr 160165 170 175 gga gaa gag cag cac act gtg gac aag ctg gtc aac atg aca tatatt 755 Gly Glu Glu Gln His Thr Val Asp Lys Leu Val Asn Met Thr Tyr Ile180 185 190 ttt caa aaa ctt gct gca gtc aaa gat caa aga gaa tgg gtc accaca 803 Phe Gln Lys Leu Ala Ala Val Lys Asp Gln Arg Glu Trp Val Thr Thr195 200 205 agt gga gcc cac aag aca tta gta aat tta ctt ggt gcc cga gatact 851 Ser Gly Ala His Lys Thr Leu Val Asn Leu Leu Gly Ala Arg Asp Thr210 215 220 aat gtt cta ttg ggt tcc ctt ctg gct ctg gct agt tta gca gaaagt 899 Asn Val Leu Leu Gly Ser Leu Leu Ala Leu Ala Ser Leu Ala Glu Ser225 230 235 caa gaa tgt agg gag aag ata agt gaa ctc aac att gta gaa aatctg 947 Gln Glu Cys Arg Glu Lys Ile Ser Glu Leu Asn Ile Val Glu Asn Leu240 245 250 255 ttg atg att tta cat gaa tat gac ttg ctt tct aaa aga ctaaca gcg 995 Leu Met Ile Leu His Glu Tyr Asp Leu Leu Ser Lys Arg Leu ThrAla 260 265 270 gag ttg ctg cgc cta ctt tgt gca gag ccc cag gtg aaa gagcag gtg 1043 Glu Leu Leu Arg Leu Leu Cys Ala Glu Pro Gln Val Lys Glu GlnVal 275 280 285 aag ctc tat gag ggg ata ccg gtc ctc ctc agt ctg ctc cactct gac 1091 Lys Leu Tyr Glu Gly Ile Pro Val Leu Leu Ser Leu Leu His SerAsp 290 295 300 cac ttg aag ctc ctc tgg agc att gtc tgg att ctg gta caggtt tgt 1139 His Leu Lys Leu Leu Trp Ser Ile Val Trp Ile Leu Val Gln ValCys 305 310 315 gag gac cct gag acc agc gtg gaa att cgc att tgg gga ggcatc aaa 1187 Glu Asp Pro Glu Thr Ser Val Glu Ile Arg Ile Trp Gly Gly IleLys 320 325 330 335 cag ctt ctt cat att tta caa gga gac aga aat ttt gtttct gat cac 1235 Gln Leu Leu His Ile Leu Gln Gly Asp Arg Asn Phe Val SerAsp His 340 345 350 tcc tcc att gga agc ctg tcc agt gca aat gct gca ggccga atc cag 1283 Ser Ser Ile Gly Ser Leu Ser Ser Ala Asn Ala Ala Gly ArgIle Gln 355 360 365 cag ctt cat tta tca gaa gac ttg agc cct agg gaa atacaa gaa aat 1331 Gln Leu His Leu Ser Glu Asp Leu Ser Pro Arg Glu Ile GlnGlu Asn 370 375 380 act ttc tca ctt caa gca gcc tgc tgt gct gcc ctc actgag ctg gtg 1379 Thr Phe Ser Leu Gln Ala Ala Cys Cys Ala Ala Leu Thr GluLeu Val 385 390 395 ctc aat gac acc aat gcc cac cag gtg gtt cag gaa aatggt gta tat 1427 Leu Asn Asp Thr Asn Ala His Gln Val Val Gln Glu Asn GlyVal Tyr 400 405 410 415 aca ata gca aaa tta att tta cca aat aag caa aagaat gca gca aaa 1475 Thr Ile Ala Lys Leu Ile Leu Pro Asn Lys Gln Lys AsnAla Ala Lys 420 425 430 agt aat cta tta cag tgt tat gct ttc aga gcc ttgaga ttt ctc ttc 1523 Ser Asn Leu Leu Gln Cys Tyr Ala Phe Arg Ala Leu ArgPhe Leu Phe 435 440 445 agt atg gaa aga aac aga cca ctc ttt aaa aga cttttc ccc aca gac 1571 Ser Met Glu Arg Asn Arg Pro Leu Phe Lys Arg Leu PhePro Thr Asp 450 455 460 ttg ttt gag atc ttc att gac ata ggg cat tat gtacgt gat atc agt 1619 Leu Phe Glu Ile Phe Ile Asp Ile Gly His Tyr Val ArgAsp Ile Ser 465 470 475 gct tat gaa gaa ttg gta tcc aag ctg aat tta ttagtg gag gat gaa 1667 Ala Tyr Glu Glu Leu Val Ser Lys Leu Asn Leu Leu ValGlu Asp Glu 480 485 490 495 ctg aag caa att gct gaa aat att gaa agc attaat cag aac aaa gct 1715 Leu Lys Gln Ile Ala Glu Asn Ile Glu Ser Ile AsnGln Asn Lys Ala 500 505 510 cct tcg aaa tat ata ggc aac tat gca att ttggat cat ctt gga agt 1763 Pro Ser Lys Tyr Ile Gly Asn Tyr Ala Ile Leu AspHis Leu Gly Ser 515 520 525 gga gct ttt ggc tgt gtt tac aag gtt aga aagcat agt ggt caa aat 1811 Gly Ala Phe Gly Cys Val Tyr Lys Val Arg Lys HisSer Gly Gln Asn 530 535 540 ctt tta gca atg aaa gag gtc aat tta cat aaccca gca ttt gga aag 1859 Leu Leu Ala Met Lys Glu Val Asn Leu His Asn ProAla Phe Gly Lys 545 550 555 gat aag aaa gat cga gac agc agc gta agg aatatt gtt tct gaa tta 1907 Asp Lys Lys Asp Arg Asp Ser Ser Val Arg Asn IleVal Ser Glu Leu 560 565 570 575 aca ata att aaa gag cag ctt tat cat cccaac att gta cgt tat tac 1955 Thr Ile Ile Lys Glu Gln Leu Tyr His Pro AsnIle Val Arg Tyr Tyr 580 585 590 aaa aca ttt ctg gaa aac gat agg ttg tacata gtt atg gag ctg ata 2003 Lys Thr Phe Leu Glu Asn Asp Arg Leu Tyr IleVal Met Glu Leu Ile 595 600 605 gaa gga gcc ccg ctt gga gag cat ttc agttct ttg aag gaa aaa cat 2051 Glu Gly Ala Pro Leu Gly Glu His Phe Ser SerLeu Lys Glu Lys His 610 615 620 cac cat ttt act gaa gaa aga cta tgg aaaata ttt ata cag ctg tgc 2099 His His Phe Thr Glu Glu Arg Leu Trp Lys IlePhe Ile Gln Leu Cys 625 630 635 tta gct ctt cga tac tta cac aag gag aagagg att gtc cat aga gat 2147 Leu Ala Leu Arg Tyr Leu His Lys Glu Lys ArgIle Val His Arg Asp 640 645 650 655 ctg aca cca aac aac att atg ttg ggggat aag gac aaa gta aca gtt 2195 Leu Thr Pro Asn Asn Ile Met Leu Gly AspLys Asp Lys Val Thr Val 660 665 670 act gac ttt ggc ctg gca aag caa aaacaa gaa aac agt aaa ctc acg 2243 Thr Asp Phe Gly Leu Ala Lys Gln Lys GlnGlu Asn Ser Lys Leu Thr 675 680 685 tct gtg gtt gga aca atc ctg tat tcttgc ccc gag gta ctg aag agt 2291 Ser Val Val Gly Thr Ile Leu Tyr Ser CysPro Glu Val Leu Lys Ser 690 695 700 gag ccg tat ggg gag aag gct gat gtctgg gca gta ggc tgc atc ctt 2339 Glu Pro Tyr Gly Glu Lys Ala Asp Val TrpAla Val Gly Cys Ile Leu 705 710 715 tat cag atg gcg act ttg agt ccc cccttc tac agc act aac atg ctg 2387 Tyr Gln Met Ala Thr Leu Ser Pro Pro PheTyr Ser Thr Asn Met Leu 720 725 730 735 tcc ttg gct aca aaa ata gtg gaggcg gta tat gaa cca gtg cca gaa 2435 Ser Leu Ala Thr Lys Ile Val Glu AlaVal Tyr Glu Pro Val Pro Glu 740 745 750 ggt atc tac tct gaa aaa gta acagac acc atc agc agg tgc ctc act 2483 Gly Ile Tyr Ser Glu Lys Val Thr AspThr Ile Ser Arg Cys Leu Thr 755 760 765 cct gat gcg gaa gct cgt cca gatatt gta gaa gtc agt tcg atg ata 2531 Pro Asp Ala Glu Ala Arg Pro Asp IleVal Glu Val Ser Ser Met Ile 770 775 780 tca gat gtc atg atg aaa tat ttagac aac tta tct aca tcc cag ttg 2579 Ser Asp Val Met Met Lys Tyr Leu AspAsn Leu Ser Thr Ser Gln Leu 785 790 795 tcc ttg gaa aag aag cta gaa cgggaa cga aga cgc aca caa agg tat 2627 Ser Leu Glu Lys Lys Leu Glu Arg GluArg Arg Arg Thr Gln Arg Tyr 800 805 810 815 ttt atg gaa gcc aac cgg aacacc gtc aca tgt cac cat gag ctg gct 2675 Phe Met Glu Ala Asn Arg Asn ThrVal Thr Cys His His Glu Leu Ala 820 825 830 gtt cta tct cac gag acc tttgag aag gca agt ttg agt agc agc agc 2723 Val Leu Ser His Glu Thr Phe GluLys Ala Ser Leu Ser Ser Ser Ser 835 840 845 agt gga gca gcc agc ctg aaaagt gaa ctt tca gaa agc gca gac ctg 2771 Ser Gly Ala Ala Ser Leu Lys SerGlu Leu Ser Glu Ser Ala Asp Leu 850 855 860 ccc cct gaa ggc ttc cag gcctcc tat ggt aaa gac gaa gac agg gcc 2819 Pro Pro Glu Gly Phe Gln Ala SerTyr Gly Lys Asp Glu Asp Arg Ala 865 870 875 tgt gac gaa atc ctg tca gatgat aac ttc aac ctg gaa aat gct gag 2867 Cys Asp Glu Ile Leu Ser Asp AspAsn Phe Asn Leu Glu Asn Ala Glu 880 885 890 895 aaa gat aca tat tca gaggta gat gat gaa ttg gac att tcg gat aac 2915 Lys Asp Thr Tyr Ser Glu ValAsp Asp Glu Leu Asp Ile Ser Asp Asn 900 905 910 tcc agc agc tcc agt tcaagc cct ctg aaa gaa tct aca ttc aac att 2963 Ser Ser Ser Ser Ser Ser SerPro Leu Lys Glu Ser Thr Phe Asn Ile 915 920 925 tta aag aga agt ttt agtgct tca gga gga gaa aga caa tcc caa aca 3011 Leu Lys Arg Ser Phe Ser AlaSer Gly Gly Glu Arg Gln Ser Gln Thr 930 935 940 agg gac ttc act gga ggaaca gga tca aga cca aga cca gca tca gca 3059 Arg Asp Phe Thr Gly Gly ThrGly Ser Arg Pro Arg Pro Ala Ser Ala 945 950 955 gga att gct gtg tcc cagagg aaa gtg cgt cag atc agt gat cct att 3107 Gly Ile Ala Val Ser Gln ArgLys Val Arg Gln Ile Ser Asp Pro Ile 960 965 970 975 cag cag ata tta attcag ctg cac aaa ata atc tat atc aca cag ctt 3155 Gln Gln Ile Leu Ile GlnLeu His Lys Ile Ile Tyr Ile Thr Gln Leu 980 985 990 cct cca gct ttg caccac aat ttg aaa aga agg gtt ata gag aga ttc 3203 Pro Pro Ala Leu His HisAsn Leu Lys Arg Arg Val Ile Glu Arg Phe 995 1000 1005 aag aaa tcc ctcttc agc cag cag agt aac cct tgt aat ttg aaa tct 3251 Lys Lys Ser Leu PheSer Gln Gln Ser Asn Pro Cys Asn Leu Lys Ser 1010 1015 1020 gaa att aaaaag tta tct cag gga tct cca gaa ccg att gag ccc aac 3299 Glu Ile Lys LysLeu Ser Gln Gly Ser Pro Glu Pro Ile Glu Pro Asn 1025 1030 1035 ttt ttcaca gca gat tac cat tta tta cat cgt tca tcc ggt gga aac 3347 Phe Phe ThrAla Asp Tyr His Leu Leu His Arg Ser Ser Gly Gly Asn 1040 1045 1050 1055agc ctg tcc cca aat gac cct aca ggt tta cca acc agc att gaa ttg 3395 SerLeu Ser Pro Asn Asp Pro Thr Gly Leu Pro Thr Ser Ile Glu Leu 1060 10651070 gag gaa gga ata aca tat gaa cag atg cag act gtg att gaa gaa gtc3443 Glu Glu Gly Ile Thr Tyr Glu Gln Met Gln Thr Val Ile Glu Glu Val1075 1080 1085 ctt gag gaa agt ggc tat tac aat ttt aca tct aac agg tatcat tcc 3491 Leu Glu Glu Ser Gly Tyr Tyr Asn Phe Thr Ser Asn Arg Tyr HisSer 1090 1095 1100 tat cca tgg ggg acc aag aat cac cca acc aaa aga tgaaaatgctgca 3540 Tyr Pro Trp Gly Thr Lys Asn His Pro Thr Lys Arg * 11051110 1115 ttttgagtgg acttgatttt ctcagtgaag ttcaagttct ggacttcagccgctattgca 3600 agatgcccaa ggattgggtg ctgctagagg gtgtggaaaa gaccaagatgccatggggcc 3660 tgcaggactt ctttctgggg gtcctgtgct ggagtatatg acagctgcggtacttgaggg 3720 cttcattgcc agaacacatt atatacagga tgtcagagct accagtgtgctgctgggaga 3780 aaatgctgca aaattcatct tttggagggt ggggggaaaa cccaaaaacaacaacaaaaa 3840 aactctctta cagaattttc cttaacatta aaaaaaactt gtcatatttttcaaaggcnc 3900 atttgatact cagaattgc 3919 2 1115 PRT Homo sapiens 2 MetPro Asp Gln Asp Lys Lys Val Lys Thr Thr Glu Lys Ser Thr Asp 1 5 10 15Lys Gln Gln Glu Ile Thr Ile Arg Asp Tyr Ser Asp Leu Lys Arg Leu 20 25 30Arg Cys Leu Leu Asn Val Gln Ser Ser Lys Gln Gln Leu Pro Ala Ile 35 40 45Asn Phe Asp Ser Ala Gln Asn Ser Met Thr Lys Ser Glu Pro Ala Ile 50 55 60Arg Ala Gly Gly His Arg Ala Arg Gly Gln Trp His Glu Ser Thr Glu 65 70 7580 Ala Val Glu Leu Glu Asn Phe Ser Ile Asn Tyr Lys Asn Glu Arg Asn 85 9095 Phe Ser Lys His Pro Gln Arg Lys Leu Phe Gln Glu Ile Phe Thr Ala 100105 110 Leu Val Lys Asn Arg Leu Ile Ser Arg Glu Trp Val Asn Arg Ala Pro115 120 125 Ser Ile His Phe Leu Arg Val Leu Ile Cys Leu Arg Leu Leu MetArg 130 135 140 Asp Pro Cys Tyr Gln Glu Ile Leu His Ser Leu Gly Gly IleGlu Asn 145 150 155 160 Leu Ala Gln Tyr Met Glu Ile Val Ala Asn Glu TyrLeu Gly Tyr Gly 165 170 175 Glu Glu Gln His Thr Val Asp Lys Leu Val AsnMet Thr Tyr Ile Phe 180 185 190 Gln Lys Leu Ala Ala Val Lys Asp Gln ArgGlu Trp Val Thr Thr Ser 195 200 205 Gly Ala His Lys Thr Leu Val Asn LeuLeu Gly Ala Arg Asp Thr Asn 210 215 220 Val Leu Leu Gly Ser Leu Leu AlaLeu Ala Ser Leu Ala Glu Ser Gln 225 230 235 240 Glu Cys Arg Glu Lys IleSer Glu Leu Asn Ile Val Glu Asn Leu Leu 245 250 255 Met Ile Leu His GluTyr Asp Leu Leu Ser Lys Arg Leu Thr Ala Glu 260 265 270 Leu Leu Arg LeuLeu Cys Ala Glu Pro Gln Val Lys Glu Gln Val Lys 275 280 285 Leu Tyr GluGly Ile Pro Val Leu Leu Ser Leu Leu His Ser Asp His 290 295 300 Leu LysLeu Leu Trp Ser Ile Val Trp Ile Leu Val Gln Val Cys Glu 305 310 315 320Asp Pro Glu Thr Ser Val Glu Ile Arg Ile Trp Gly Gly Ile Lys Gln 325 330335 Leu Leu His Ile Leu Gln Gly Asp Arg Asn Phe Val Ser Asp His Ser 340345 350 Ser Ile Gly Ser Leu Ser Ser Ala Asn Ala Ala Gly Arg Ile Gln Gln355 360 365 Leu His Leu Ser Glu Asp Leu Ser Pro Arg Glu Ile Gln Glu AsnThr 370 375 380 Phe Ser Leu Gln Ala Ala Cys Cys Ala Ala Leu Thr Glu LeuVal Leu 385 390 395 400 Asn Asp Thr Asn Ala His Gln Val Val Gln Glu AsnGly Val Tyr Thr 405 410 415 Ile Ala Lys Leu Ile Leu Pro Asn Lys Gln LysAsn Ala Ala Lys Ser 420 425 430 Asn Leu Leu Gln Cys Tyr Ala Phe Arg AlaLeu Arg Phe Leu Phe Ser 435 440 445 Met Glu Arg Asn Arg Pro Leu Phe LysArg Leu Phe Pro Thr Asp Leu 450 455 460 Phe Glu Ile Phe Ile Asp Ile GlyHis Tyr Val Arg Asp Ile Ser Ala 465 470 475 480 Tyr Glu Glu Leu Val SerLys Leu Asn Leu Leu Val Glu Asp Glu Leu 485 490 495 Lys Gln Ile Ala GluAsn Ile Glu Ser Ile Asn Gln Asn Lys Ala Pro 500 505 510 Ser Lys Tyr IleGly Asn Tyr Ala Ile Leu Asp His Leu Gly Ser Gly 515 520 525 Ala Phe GlyCys Val Tyr Lys Val Arg Lys His Ser Gly Gln Asn Leu 530 535 540 Leu AlaMet Lys Glu Val Asn Leu His Asn Pro Ala Phe Gly Lys Asp 545 550 555 560Lys Lys Asp Arg Asp Ser Ser Val Arg Asn Ile Val Ser Glu Leu Thr 565 570575 Ile Ile Lys Glu Gln Leu Tyr His Pro Asn Ile Val Arg Tyr Tyr Lys 580585 590 Thr Phe Leu Glu Asn Asp Arg Leu Tyr Ile Val Met Glu Leu Ile Glu595 600 605 Gly Ala Pro Leu Gly Glu His Phe Ser Ser Leu Lys Glu Lys HisHis 610 615 620 His Phe Thr Glu Glu Arg Leu Trp Lys Ile Phe Ile Gln LeuCys Leu 625 630 635 640 Ala Leu Arg Tyr Leu His Lys Glu Lys Arg Ile ValHis Arg Asp Leu 645 650 655 Thr Pro Asn Asn Ile Met Leu Gly Asp Lys AspLys Val Thr Val Thr 660 665 670 Asp Phe Gly Leu Ala Lys Gln Lys Gln GluAsn Ser Lys Leu Thr Ser 675 680 685 Val Val Gly Thr Ile Leu Tyr Ser CysPro Glu Val Leu Lys Ser Glu 690 695 700 Pro Tyr Gly Glu Lys Ala Asp ValTrp Ala Val Gly Cys Ile Leu Tyr 705 710 715 720 Gln Met Ala Thr Leu SerPro Pro Phe Tyr Ser Thr Asn Met Leu Ser 725 730 735 Leu Ala Thr Lys IleVal Glu Ala Val Tyr Glu Pro Val Pro Glu Gly 740 745 750 Ile Tyr Ser GluLys Val Thr Asp Thr Ile Ser Arg Cys Leu Thr Pro 755 760 765 Asp Ala GluAla Arg Pro Asp Ile Val Glu Val Ser Ser Met Ile Ser 770 775 780 Asp ValMet Met Lys Tyr Leu Asp Asn Leu Ser Thr Ser Gln Leu Ser 785 790 795 800Leu Glu Lys Lys Leu Glu Arg Glu Arg Arg Arg Thr Gln Arg Tyr Phe 805 810815 Met Glu Ala Asn Arg Asn Thr Val Thr Cys His His Glu Leu Ala Val 820825 830 Leu Ser His Glu Thr Phe Glu Lys Ala Ser Leu Ser Ser Ser Ser Ser835 840 845 Gly Ala Ala Ser Leu Lys Ser Glu Leu Ser Glu Ser Ala Asp LeuPro 850 855 860 Pro Glu Gly Phe Gln Ala Ser Tyr Gly Lys Asp Glu Asp ArgAla Cys 865 870 875 880 Asp Glu Ile Leu Ser Asp Asp Asn Phe Asn Leu GluAsn Ala Glu Lys 885 890 895 Asp Thr Tyr Ser Glu Val Asp Asp Glu Leu AspIle Ser Asp Asn Ser 900 905 910 Ser Ser Ser Ser Ser Ser Pro Leu Lys GluSer Thr Phe Asn Ile Leu 915 920 925 Lys Arg Ser Phe Ser Ala Ser Gly GlyGlu Arg Gln Ser Gln Thr Arg 930 935 940 Asp Phe Thr Gly Gly Thr Gly SerArg Pro Arg Pro Ala Ser Ala Gly 945 950 955 960 Ile Ala Val Ser Gln ArgLys Val Arg Gln Ile Ser Asp Pro Ile Gln 965 970 975 Gln Ile Leu Ile GlnLeu His Lys Ile Ile Tyr Ile Thr Gln Leu Pro 980 985 990 Pro Ala Leu HisHis Asn Leu Lys Arg Arg Val Ile Glu Arg Phe Lys 995 1000 1005 Lys SerLeu Phe Ser Gln Gln Ser Asn Pro Cys Asn Leu Lys Ser Glu 1010 1015 1020Ile Lys Lys Leu Ser Gln Gly Ser Pro Glu Pro Ile Glu Pro Asn Phe 10251030 1035 1040 Phe Thr Ala Asp Tyr His Leu Leu His Arg Ser Ser Gly GlyAsn Ser 1045 1050 1055 Leu Ser Pro Asn Asp Pro Thr Gly Leu Pro Thr SerIle Glu Leu Glu 1060 1065 1070 Glu Gly Ile Thr Tyr Glu Gln Met Gln ThrVal Ile Glu Glu Val Leu 1075 1080 1085 Glu Glu Ser Gly Tyr Tyr Asn PheThr Ser Asn Arg Tyr His Ser Tyr 1090 1095 1100 Pro Trp Gly Thr Lys AsnHis Pro Thr Lys Arg 1105 1110 1115 3 3348 DNA Homo sapiens CDS(1)...(3348) 3 atg cct gat caa gat aaa aag gtg aag acc aca gaa aaa tcaact gat 48 Met Pro Asp Gln Asp Lys Lys Val Lys Thr Thr Glu Lys Ser ThrAsp 1 5 10 15 aaa cag caa gaa atc acc atc agg gac tat tca gat ctt aaaaga ctt 96 Lys Gln Gln Glu Ile Thr Ile Arg Asp Tyr Ser Asp Leu Lys ArgLeu 20 25 30 cgg tgc ctt ttg aac gtc caa tca agc aaa caa cag ctt cca gccatt 144 Arg Cys Leu Leu Asn Val Gln Ser Ser Lys Gln Gln Leu Pro Ala Ile35 40 45 aac ttc gat agt gcc caa aat agc atg acg aag tct gag ccc gcc atc192 Asn Phe Asp Ser Ala Gln Asn Ser Met Thr Lys Ser Glu Pro Ala Ile 5055 60 agg gcg ggt gga cac aga gct cgg ggt cag tgg cat gaa tcc aca gaa240 Arg Ala Gly Gly His Arg Ala Arg Gly Gln Trp His Glu Ser Thr Glu 6570 75 80 gct gtt gaa ctt gaa aat ttt agt ata aac tac aag aat gag aga aat288 Ala Val Glu Leu Glu Asn Phe Ser Ile Asn Tyr Lys Asn Glu Arg Asn 8590 95 ttc agc aaa cat cct cag cgt aaa cta ttt cag gag atc ttt acc gcc336 Phe Ser Lys His Pro Gln Arg Lys Leu Phe Gln Glu Ile Phe Thr Ala 100105 110 ttg gtg aaa aat aga ctc ata agc aga gag tgg gtt aat cga gcc cca384 Leu Val Lys Asn Arg Leu Ile Ser Arg Glu Trp Val Asn Arg Ala Pro 115120 125 tct att cat ttt ctg aga gtg tta atc tgt ctg agg cta cta atg agg432 Ser Ile His Phe Leu Arg Val Leu Ile Cys Leu Arg Leu Leu Met Arg 130135 140 gat cca tgt tat cag gaa ata ctc cat agc ttg ggt ggg att gaa aac480 Asp Pro Cys Tyr Gln Glu Ile Leu His Ser Leu Gly Gly Ile Glu Asn 145150 155 160 cta gct cag tat atg gag att gta gcc aat gag tac ctc ggc tatgga 528 Leu Ala Gln Tyr Met Glu Ile Val Ala Asn Glu Tyr Leu Gly Tyr Gly165 170 175 gaa gag cag cac act gtg gac aag ctg gtc aac atg aca tat attttt 576 Glu Glu Gln His Thr Val Asp Lys Leu Val Asn Met Thr Tyr Ile Phe180 185 190 caa aaa ctt gct gca gtc aaa gat caa aga gaa tgg gtc acc acaagt 624 Gln Lys Leu Ala Ala Val Lys Asp Gln Arg Glu Trp Val Thr Thr Ser195 200 205 gga gcc cac aag aca tta gta aat tta ctt ggt gcc cga gat actaat 672 Gly Ala His Lys Thr Leu Val Asn Leu Leu Gly Ala Arg Asp Thr Asn210 215 220 gtt cta ttg ggt tcc ctt ctg gct ctg gct agt tta gca gaa agtcaa 720 Val Leu Leu Gly Ser Leu Leu Ala Leu Ala Ser Leu Ala Glu Ser Gln225 230 235 240 gaa tgt agg gag aag ata agt gaa ctc aac att gta gaa aatctg ttg 768 Glu Cys Arg Glu Lys Ile Ser Glu Leu Asn Ile Val Glu Asn LeuLeu 245 250 255 atg att tta cat gaa tat gac ttg ctt tct aaa aga cta acagcg gag 816 Met Ile Leu His Glu Tyr Asp Leu Leu Ser Lys Arg Leu Thr AlaGlu 260 265 270 ttg ctg cgc cta ctt tgt gca gag ccc cag gtg aaa gag caggtg aag 864 Leu Leu Arg Leu Leu Cys Ala Glu Pro Gln Val Lys Glu Gln ValLys 275 280 285 ctc tat gag ggg ata ccg gtc ctc ctc agt ctg ctc cac tctgac cac 912 Leu Tyr Glu Gly Ile Pro Val Leu Leu Ser Leu Leu His Ser AspHis 290 295 300 ttg aag ctc ctc tgg agc att gtc tgg att ctg gta cag gtttgt gag 960 Leu Lys Leu Leu Trp Ser Ile Val Trp Ile Leu Val Gln Val CysGlu 305 310 315 320 gac cct gag acc agc gtg gaa att cgc att tgg gga ggcatc aaa cag 1008 Asp Pro Glu Thr Ser Val Glu Ile Arg Ile Trp Gly Gly IleLys Gln 325 330 335 ctt ctt cat att tta caa gga gac aga aat ttt gtt tctgat cac tcc 1056 Leu Leu His Ile Leu Gln Gly Asp Arg Asn Phe Val Ser AspHis Ser 340 345 350 tcc att gga agc ctg tcc agt gca aat gct gca ggc cgaatc cag cag 1104 Ser Ile Gly Ser Leu Ser Ser Ala Asn Ala Ala Gly Arg IleGln Gln 355 360 365 ctt cat tta tca gaa gac ttg agc cct agg gaa ata caagaa aat act 1152 Leu His Leu Ser Glu Asp Leu Ser Pro Arg Glu Ile Gln GluAsn Thr 370 375 380 ttc tca ctt caa gca gcc tgc tgt gct gcc ctc act gagctg gtg ctc 1200 Phe Ser Leu Gln Ala Ala Cys Cys Ala Ala Leu Thr Glu LeuVal Leu 385 390 395 400 aat gac acc aat gcc cac cag gtg gtt cag gaa aatggt gta tat aca 1248 Asn Asp Thr Asn Ala His Gln Val Val Gln Glu Asn GlyVal Tyr Thr 405 410 415 ata gca aaa tta att tta cca aat aag caa aag aatgca gca aaa agt 1296 Ile Ala Lys Leu Ile Leu Pro Asn Lys Gln Lys Asn AlaAla Lys Ser 420 425 430 aat cta tta cag tgt tat gct ttc aga gcc ttg agattt ctc ttc agt 1344 Asn Leu Leu Gln Cys Tyr Ala Phe Arg Ala Leu Arg PheLeu Phe Ser 435 440 445 atg gaa aga aac aga cca ctc ttt aaa aga ctt ttcccc aca gac ttg 1392 Met Glu Arg Asn Arg Pro Leu Phe Lys Arg Leu Phe ProThr Asp Leu 450 455 460 ttt gag atc ttc att gac ata ggg cat tat gta cgtgat atc agt gct 1440 Phe Glu Ile Phe Ile Asp Ile Gly His Tyr Val Arg AspIle Ser Ala 465 470 475 480 tat gaa gaa ttg gta tcc aag ctg aat tta ttagtg gag gat gaa ctg 1488 Tyr Glu Glu Leu Val Ser Lys Leu Asn Leu Leu ValGlu Asp Glu Leu 485 490 495 aag caa att gct gaa aat att gaa agc att aatcag aac aaa gct cct 1536 Lys Gln Ile Ala Glu Asn Ile Glu Ser Ile Asn GlnAsn Lys Ala Pro 500 505 510 tcg aaa tat ata ggc aac tat gca att ttg gatcat ctt gga agt gga 1584 Ser Lys Tyr Ile Gly Asn Tyr Ala Ile Leu Asp HisLeu Gly Ser Gly 515 520 525 gct ttt ggc tgt gtt tac aag gtt aga aag catagt ggt caa aat ctt 1632 Ala Phe Gly Cys Val Tyr Lys Val Arg Lys His SerGly Gln Asn Leu 530 535 540 tta gca atg aaa gag gtc aat tta cat aac ccagca ttt gga aag gat 1680 Leu Ala Met Lys Glu Val Asn Leu His Asn Pro AlaPhe Gly Lys Asp 545 550 555 560 aag aaa gat cga gac agc agc gta agg aatatt gtt tct gaa tta aca 1728 Lys Lys Asp Arg Asp Ser Ser Val Arg Asn IleVal Ser Glu Leu Thr 565 570 575 ata att aaa gag cag ctt tat cat ccc aacatt gta cgt tat tac aaa 1776 Ile Ile Lys Glu Gln Leu Tyr His Pro Asn IleVal Arg Tyr Tyr Lys 580 585 590 aca ttt ctg gaa aac gat agg ttg tac atagtt atg gag ctg ata gaa 1824 Thr Phe Leu Glu Asn Asp Arg Leu Tyr Ile ValMet Glu Leu Ile Glu 595 600 605 gga gcc ccg ctt gga gag cat ttc agt tctttg aag gaa aaa cat cac 1872 Gly Ala Pro Leu Gly Glu His Phe Ser Ser LeuLys Glu Lys His His 610 615 620 cat ttt act gaa gaa aga cta tgg aaa atattt ata cag ctg tgc tta 1920 His Phe Thr Glu Glu Arg Leu Trp Lys Ile PheIle Gln Leu Cys Leu 625 630 635 640 gct ctt cga tac tta cac aag gag aagagg att gtc cat aga gat ctg 1968 Ala Leu Arg Tyr Leu His Lys Glu Lys ArgIle Val His Arg Asp Leu 645 650 655 aca cca aac aac att atg ttg ggg gataag gac aaa gta aca gtt act 2016 Thr Pro Asn Asn Ile Met Leu Gly Asp LysAsp Lys Val Thr Val Thr 660 665 670 gac ttt ggc ctg gca aag caa aaa caagaa aac agt aaa ctc acg tct 2064 Asp Phe Gly Leu Ala Lys Gln Lys Gln GluAsn Ser Lys Leu Thr Ser 675 680 685 gtg gtt gga aca atc ctg tat tct tgcccc gag gta ctg aag agt gag 2112 Val Val Gly Thr Ile Leu Tyr Ser Cys ProGlu Val Leu Lys Ser Glu 690 695 700 ccg tat ggg gag aag gct gat gtc tgggca gta ggc tgc atc ctt tat 2160 Pro Tyr Gly Glu Lys Ala Asp Val Trp AlaVal Gly Cys Ile Leu Tyr 705 710 715 720 cag atg gcg act ttg agt ccc cccttc tac agc act aac atg ctg tcc 2208 Gln Met Ala Thr Leu Ser Pro Pro PheTyr Ser Thr Asn Met Leu Ser 725 730 735 ttg gct aca aaa ata gtg gag gcggta tat gaa cca gtg cca gaa ggt 2256 Leu Ala Thr Lys Ile Val Glu Ala ValTyr Glu Pro Val Pro Glu Gly 740 745 750 atc tac tct gaa aaa gta aca gacacc atc agc agg tgc ctc act cct 2304 Ile Tyr Ser Glu Lys Val Thr Asp ThrIle Ser Arg Cys Leu Thr Pro 755 760 765 gat gcg gaa gct cgt cca gat attgta gaa gtc agt tcg atg ata tca 2352 Asp Ala Glu Ala Arg Pro Asp Ile ValGlu Val Ser Ser Met Ile Ser 770 775 780 gat gtc atg atg aaa tat tta gacaac tta tct aca tcc cag ttg tcc 2400 Asp Val Met Met Lys Tyr Leu Asp AsnLeu Ser Thr Ser Gln Leu Ser 785 790 795 800 ttg gaa aag aag cta gaa cgggaa cga aga cgc aca caa agg tat ttt 2448 Leu Glu Lys Lys Leu Glu Arg GluArg Arg Arg Thr Gln Arg Tyr Phe 805 810 815 atg gaa gcc aac cgg aac accgtc aca tgt cac cat gag ctg gct gtt 2496 Met Glu Ala Asn Arg Asn Thr ValThr Cys His His Glu Leu Ala Val 820 825 830 cta tct cac gag acc ttt gagaag gca agt ttg agt agc agc agc agt 2544 Leu Ser His Glu Thr Phe Glu LysAla Ser Leu Ser Ser Ser Ser Ser 835 840 845 gga gca gcc agc ctg aaa agtgaa ctt tca gaa agc gca gac ctg ccc 2592 Gly Ala Ala Ser Leu Lys Ser GluLeu Ser Glu Ser Ala Asp Leu Pro 850 855 860 cct gaa ggc ttc cag gcc tcctat ggt aaa gac gaa gac agg gcc tgt 2640 Pro Glu Gly Phe Gln Ala Ser TyrGly Lys Asp Glu Asp Arg Ala Cys 865 870 875 880 gac gaa atc ctg tca gatgat aac ttc aac ctg gaa aat gct gag aaa 2688 Asp Glu Ile Leu Ser Asp AspAsn Phe Asn Leu Glu Asn Ala Glu Lys 885 890 895 gat aca tat tca gag gtagat gat gaa ttg gac att tcg gat aac tcc 2736 Asp Thr Tyr Ser Glu Val AspAsp Glu Leu Asp Ile Ser Asp Asn Ser 900 905 910 agc agc tcc agt tca agccct ctg aaa gaa tct aca ttc aac att tta 2784 Ser Ser Ser Ser Ser Ser ProLeu Lys Glu Ser Thr Phe Asn Ile Leu 915 920 925 aag aga agt ttt agt gcttca gga gga gaa aga caa tcc caa aca agg 2832 Lys Arg Ser Phe Ser Ala SerGly Gly Glu Arg Gln Ser Gln Thr Arg 930 935 940 gac ttc act gga gga acagga tca aga cca aga cca gca tca gca gga 2880 Asp Phe Thr Gly Gly Thr GlySer Arg Pro Arg Pro Ala Ser Ala Gly 945 950 955 960 att gct gtg tcc cagagg aaa gtg cgt cag atc agt gat cct att cag 2928 Ile Ala Val Ser Gln ArgLys Val Arg Gln Ile Ser Asp Pro Ile Gln 965 970 975 cag ata tta att cagctg cac aaa ata atc tat atc aca cag ctt cct 2976 Gln Ile Leu Ile Gln LeuHis Lys Ile Ile Tyr Ile Thr Gln Leu Pro 980 985 990 cca gct ttg cac cacaat ttg aaa aga agg gtt ata gag aga ttc aag 3024 Pro Ala Leu His His AsnLeu Lys Arg Arg Val Ile Glu Arg Phe Lys 995 1000 1005 aaa tcc ctc ttcagc cag cag agt aac cct tgt aat ttg aaa tct gaa 3072 Lys Ser Leu Phe SerGln Gln Ser Asn Pro Cys Asn Leu Lys Ser Glu 1010 1015 1020 att aaa aagtta tct cag gga tct cca gaa ccg att gag ccc aac ttt 3120 Ile Lys Lys LeuSer Gln Gly Ser Pro Glu Pro Ile Glu Pro Asn Phe 1025 1030 1035 1040 ttcaca gca gat tac cat tta tta cat cgt tca tcc ggt gga aac agc 3168 Phe ThrAla Asp Tyr His Leu Leu His Arg Ser Ser Gly Gly Asn Ser 1045 1050 1055ctg tcc cca aat gac cct aca ggt tta cca acc agc att gaa ttg gag 3216 LeuSer Pro Asn Asp Pro Thr Gly Leu Pro Thr Ser Ile Glu Leu Glu 1060 10651070 gaa gga ata aca tat gaa cag atg cag act gtg att gaa gaa gtc ctt3264 Glu Gly Ile Thr Tyr Glu Gln Met Gln Thr Val Ile Glu Glu Val Leu1075 1080 1085 gag gaa agt ggc tat tac aat ttt aca tct aac agg tat cattcc tat 3312 Glu Glu Ser Gly Tyr Tyr Asn Phe Thr Ser Asn Arg Tyr His SerTyr 1090 1095 1100 cca tgg ggg acc aag aat cac cca acc aaa aga tga 3348Pro Trp Gly Thr Lys Asn His Pro Thr Lys Arg * 1105 1110 1115 4 266 PRTArtificial Sequence consensus 4 Leu Gly Glu Gly Ser Phe Gly Lys Val TyrLys Ala Lys His Lys Thr 1 5 10 15 Gly Lys Ile Val Ala Val Lys Ile LeuLys Lys Glu Ser Leu Ser Leu 20 25 30 Arg Glu Ile Gln Ile Leu Lys Arg LeuSer His Pro Asn Ile Val Arg 35 40 45 Leu Leu Gly Val Phe Glu Asp Thr AspAsp His Leu Tyr Leu Val Met 50 55 60 Glu Tyr Met Glu Gly Gly Asp Leu PheAsp Tyr Leu Arg Arg Asn Gly 65 70 75 80 Pro Leu Ser Glu Lys Glu Ala LysLys Ile Ala Leu Gln Ile Leu Arg 85 90 95 Gly Leu Glu Tyr Leu His Ser AsnGly Ile Val His Arg Asp Leu Lys 100 105 110 Pro Glu Asn Ile Leu Leu AspGlu Asn Gly Thr Val Lys Ile Ala Asp 115 120 125 Phe Gly Leu Ala Arg LeuLeu Glu Lys Leu Thr Thr Phe Val Gly Thr 130 135 140 Pro Trp Tyr Met MetAla Pro Glu Val Ile Leu Glu Gly Arg Gly Tyr 145 150 155 160 Ser Ser LysVal Asp Val Trp Ser Leu Gly Val Ile Leu Tyr Glu Leu 165 170 175 Leu ThrGly Gly Pro Leu Phe Pro Gly Ala Asp Leu Pro Ala Phe Thr 180 185 190 GlyGly Asp Glu Val Asp Gln Leu Ile Ile Phe Val Leu Lys Leu Pro 195 200 205Phe Ser Asp Glu Leu Pro Lys Thr Arg Ile Asp Pro Leu Glu Glu Leu 210 215220 Phe Arg Ile Lys Lys Arg Arg Leu Pro Leu Pro Ser Asn Cys Ser Glu 225230 235 240 Glu Leu Lys Asp Leu Leu Lys Lys Cys Leu Asn Lys Asp Pro SerLys 245 250 255 Arg Pro Gly Ser Ala Thr Ala Lys Glu Ile 260 265 5 40 PRTArtificial Sequence consensus 5 Asn Pro Glu Asn Lys Gln Ala Val Val GluAla Gly Ala Leu Pro Pro 1 5 10 15 Leu Val Gln Leu Leu Ser Pro Asp GluGlu Val Gln Glu Glu Ala Ala 20 25 30 Trp Ala Leu Ser Asn Leu Ala Ala 3540 6 34 PRT Artificial Sequence consensus 6 Xaa Gly Xaa Gly Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Lys 7 13 PRTArtificial Sequence consensus 7 Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa AsnXaa Xaa Xaa 1 5 10 8 9 PRT Artificial Sequence consensus 8 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Leu 1 5 9 854 PRT Homo sapiens 9 Met Arg Val Leu PheAsp Glu Ser Val Leu Pro Pro Thr Val Tyr Phe 1 5 10 15 Lys Asn Cys SerIle Leu Phe Leu Ala Ser Leu Cys Ala Phe Gly Val 20 25 30 Leu Thr Gly LeuLeu Val Trp Ser Phe Met Gln Tyr Met Glu Ile Val 35 40 45 Ala Asn Glu TyrLeu Gly Tyr Gly Glu Glu Gln His Thr Val Asp Lys 50 55 60 Leu Val Asn MetThr Tyr Ile Phe Gln Lys Leu Ala Ala Val Lys Asp 65 70 75 80 Gln Arg GluTrp Val Thr Thr Ser Gly Ala His Lys Thr Leu Val Asn 85 90 95 Leu Leu GlyAla Arg Asp Thr Asn Val Leu Leu Gly Ser Leu Leu Ala 100 105 110 Leu AlaSer Leu Ala Glu Arg Leu Thr Ala Glu Leu Leu Arg Leu Leu 115 120 125 CysAla Glu Pro Gln Val Lys Glu Gln Val Lys Leu Tyr Glu Gly Ile 130 135 140Pro Val Leu Leu Ser Leu Leu His Ser Asp His Leu Lys Leu Leu Trp 145 150155 160 Ser Ile Val Trp Ile Leu Val Gln Val Cys Glu Asp Pro Glu Thr Ser165 170 175 Val Glu Ile Arg Ile Trp Gly Gly Ile Lys Gln Leu Leu His IleLeu 180 185 190 Gln Gly Asp Arg Asn Phe Val Ser Asp His Ser Ser Ile GlySer Leu 195 200 205 Ser Ser Ala Asn Ala Ala Gly Arg Ile Gln Gln Leu HisLeu Ser Glu 210 215 220 Asp Leu Ser Pro Arg Glu Ile Gln Glu Asn Thr PheSer Leu Gln Ala 225 230 235 240 Ala Cys Cys Ala Ala Leu Thr Glu Leu ValLeu Asn Asp Thr Asn Ala 245 250 255 His Gln Val Val Gln Glu Asn Gly ValTyr Thr Ile Ala Lys Leu Ile 260 265 270 Leu Pro Asn Lys Gln Lys Asn AlaAla Lys Ser Asn Leu Leu Gln Cys 275 280 285 Tyr Ala Phe Arg Ala Leu ArgPhe Leu Phe Ser Met Glu Arg Asn Arg 290 295 300 Pro Leu Phe Lys Arg LeuPhe Pro Thr Asp Leu Phe Glu Ile Phe Ile 305 310 315 320 Asp Ile Gly HisTyr Val Arg Asp Ile Ser Ala Tyr Glu Glu Leu Val 325 330 335 Ser Lys LeuAsn Leu Leu Val Glu Asp Glu Leu Lys Gln Ile Ala Glu 340 345 350 Asn IleGlu Ser Ile Asn Gln Asn Lys Ala Pro Leu Lys Tyr Ile Gly 355 360 365 AsnTyr Ala Ile Leu Asp His Leu Gly Ser Gly Ala Phe Gly Cys Val 370 375 380Tyr Lys Val Arg Lys His Ser Gly Gln Asn Leu Leu Ala Met Lys Glu 385 390395 400 Val Asn Leu His Asn Pro Ala Phe Gly Lys Asp Lys Lys Asp Arg Asp405 410 415 Ser Ser Val Arg Asn Ile Val Ser Glu Leu Thr Ile Ile Lys GluGln 420 425 430 Leu Tyr His Pro Asn Ile Val Arg Tyr Tyr Lys Thr Phe LeuGlu Asn 435 440 445 Asp Arg Leu Tyr Ile Val Met Glu Leu Ile Glu Gly AlaPro Leu Gly 450 455 460 Glu His Phe Ser Ser Leu Lys Glu Lys His His HisPhe Thr Glu Glu 465 470 475 480 Arg Leu Trp Lys Ile Phe Ile Gln Leu CysLeu Ala Leu Arg Tyr Leu 485 490 495 His Lys Glu Lys Arg Ile Val His ArgAsp Gln Thr Pro Asn Asn Ile 500 505 510 Met Leu Gly Asp Lys Asp Lys ValThr Val Thr Asp Phe Gly Leu Ala 515 520 525 Lys Gln Lys Gln Glu Asn SerLys Leu Thr Ser Val Val Gly Thr Ile 530 535 540 Leu Tyr Ser Cys Pro GluVal Leu Lys Ser Glu Pro Tyr Gly Glu Lys 545 550 555 560 Ala Asp Val TrpAla Val Gly Cys Ile Leu Tyr Gln Met Ala Thr Leu 565 570 575 Ser Pro ProPhe Tyr Ser Thr Asn Met Leu Ser Leu Ala Thr Lys Ile 580 585 590 Val GluAla Val Tyr Glu Pro Val Pro Glu Gly Ile Tyr Ser Glu Lys 595 600 605 ValThr Asp Thr Ile Ser Arg Cys Leu Thr Pro Asp Ala Glu Ala Arg 610 615 620Pro Asp Ile Val Glu Val Ser Ser Met Ile Ser Asp Val Met Met Lys 625 630635 640 Tyr Leu Asp Asn Leu Ser Thr Ser Gln Leu Ser Leu Glu Lys Lys Leu645 650 655 Glu Arg Glu Arg Arg Arg Thr Gln Arg Tyr Phe Met Glu Ala AsnArg 660 665 670 Asn Thr Val Thr Cys His His Glu Leu Ala Val Leu Ser HisGlu Thr 675 680 685 Phe Glu Lys Ala Ser Leu Ser Ser Ser Ser Ser Gly AlaAla Ser Leu 690 695 700 Lys Ser Glu Leu Ser Glu Ser Ala Asp Leu Pro ProGlu Gly Phe Gln 705 710 715 720 Ala Ser Tyr Gly Lys Asp Glu Asp Arg AlaCys Asn Glu Ile Leu Ser 725 730 735 Asp Asp Asn Phe Asn Leu Glu Asn AlaGlu Lys Asp Thr Tyr Ser Glu 740 745 750 Val Asp Asp Glu Leu Asp Ile SerAsp Asn Ser Ser Ser Ser Ser Ser 755 760 765 Ser Pro Leu Lys Glu Ser ThrPhe Asn Ile Leu Lys Arg Ser Phe Ser 770 775 780 Ala Ser Gly Gly Glu ArgGln Ser Gln Thr Arg Asp Phe Thr Gly Gly 785 790 795 800 Thr Gly Ser ArgPro Arg Pro Gly Pro Gln Met Gly Thr Phe Leu Trp 805 810 815 Gln Ala SerAla Gly Ile Ala Val Ser Gln Arg Lys Val Arg Gln Ile 820 825 830 Ser AspPro Ile Gln Gln Ile Leu Ile Gln Leu His Lys Ile Ile Tyr 835 840 845 IleThr Gln Leu Pro Pro 850 10 23 DNA Artificial Sequence primer 10aagactgggc tgagtttgtg aag 23 11 21 DNA Artificial Sequence primer 11ccaagggatt gcctacaaac a 21 12 24 DNA Artificial Sequence probe 12aactgccatg cctcgaagac ctgg 24

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of: a) a nucleic acid molecule comprising anucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number ______; b) anucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC as Accession Number______; c) a nucleic acid molecule which encodes a polypeptidecomprising an amino acid sequence at least 90% identical to the aminoacid sequence of SEQ ID NO:2, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC as Accession Number______, wherein the polypeptide has kinase activity; and d) a nucleicacid molecule which encodes a polypeptide comprising an amino acidsequence at least 90% identical to the amino acid sequence of SEQ IDNO:2, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number ______, wherein thepolypeptide does not have kinase activity.
 2. The nucleic acid moleculeof claim 1 further comprising vector nucleic acid sequences.
 3. Thenucleic acid molecule of claim 1 further comprising nucleic acidsequences encoding a heterologous polypeptide.
 4. A host cell whichcontains the nucleic acid molecule of claim
 1. 5. The host cell of claim5 which is a mammalian host cell.
 6. A non-human mammalian host cellcontaining the nucleic acid molecule of claim
 1. 7. An isolatedpolypeptide selected from the group consisting of: a) a polypeptidewhich is encoded by a nucleic acid molecule comprising a nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, the amino acid sequence encoded bythe cDNA insert of the plasmid deposited with the ATCC as AccessionNumber ______, or a complement thereof; b) a polypeptide comprising theamino acid sequence of SEQ ID NO:2; c) a polypeptide comprising an aminoacid sequence which is at least 90% identical to the amino acid sequenceof SEQ ID NO:2, wherein the polypeptide has kinase activity; and d) apolypeptide comprising an amino acid sequence which is at least 90%identical to the amino acid sequence of SEQ ID NO:2, wherein thepolypeptide does not have kinase activity.
 8. The polypeptide of claim 7further comprising heterologous amino acid sequences.
 9. An antibodywhich selectively binds to a polypeptide of claim
 7. 10. A method forproducing a polypeptide selected from the group consisting of: a) apolypeptide comprising the amino acid sequence of SEQ ID NO:2, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith the ATCC as Accession Number ______; b) a polypeptide comprising anamino acid sequence which is at least 90% identical to the amino acidsequence of SEQ ID NO:2, wherein the polypeptide has kinase activity;and c) a polypeptide comprising an amino acid sequence which is at least90% identical to the amino acid sequence of SEQ ID NO:2, wherein thepolypeptide does not have kinase activity; comprising culturing the hostcell of claim 4 under conditions in which the nucleic acid molecule isexpressed.
 11. A method for detecting the presence of a polypeptide ofclaim 7 in a sample, comprising: a) contacting the sample with acompound which selectively binds to a polypeptide of claim 7; and b)determining whether the compound binds to the polypeptide in the sample.12. The method of claim 11, wherein the compound which binds to thepolypeptide is an antibody.
 13. A kit comprising a compound whichselectively binds to a polypeptide of claim 7 and instructions for use.14. A method for detecting the presence of a nucleic acid molecule ofclaim I in a sample, comprising the steps of: a) contacting the samplewith a nucleic acid probe or primer which selectively hybridizes to thenucleic acid molecule; and b) determining whether the nucleic acid probeor primer binds to a nucleic acid molecule in the sample.
 15. The methodof claim 14, wherein the sample comprises mRNA molecules and iscontacted with a nucleic acid probe.
 16. A kit comprising a compoundwhich selectively hybridizes to a nucleic acid molecule of claim 1 andinstructions for use.
 17. A method for identifying a compound whichbinds to a polypeptide of claim 7 comprising the steps of: a) contactinga polypeptide, or a cell expressing a polypeptide of claim 7 with a testcompound; and b) determining whether the polypeptide binds to the testcompound.
 18. The method of claim 17, wherein the binding of the testcompound to the polypeptide is detected by a method selected from thegroup consisting of: a) detection of binding by direct detecting of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; and c) detection of binding using an assayfor 14815 activity.
 19. A method for modulating the activity of apolypeptide of claim 7 comprising contacting a polypeptide or a cellexpressing a polypeptide of claim 7 with a compound which binds to thepolypeptide in a sufficient concentration to modulate the activity ofthe polypeptide.
 20. A method for identifying a compound which modulatesthe activity of a polypeptide of claim 7, comprising: a) contacting apolypeptide of claim 7 with a test compound; and b) determining theeffect of the test compound on the activity of the polypeptide tothereby identify a compound which modulates the activity of thepolypeptide.